Mycotaxon

MYCOTAXON

THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE

VOLUME 137 (1) JANUARY-MARCH 2022

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Claviceps bavariensis sp. nov.
(Liu, Tanaka, Kolarik— Fie. 3, p. 81)

ISSN (PRINT) 0093-4666 https://doi.org/10.5248/137-1 ISSN (ONLINE) 2154-8889
MYXNAE 137(1): 1-172 (2022)

EDITORIAL ADVISORY BOARD

ELSE C. VELLINGA (2019-2022), Chair
Berkeley, California, U.S.A.

KAREN HANSEN (2014-2021), Past Chair
Stockholm, Sweden

XINLI WEI (2019-2023)

Beijing, China

Topp W. OsMUNDSON (2019-2024)

La Crosse, Wisconsin, U.S.A.

ELAINE MALosso (2019-2025)
Recife, Brazil

ISSN 0093-4666 (PRINT)
ISSN 2154-8889 (ONLINE)

MYCOTAXON

THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE

JANUARY-MARCH 2022

VOLUME 137 (1)

http://dx.doi.org/10.5248/137-1

EDITOR-IN-CHIEF

LORELEI L. NORVELL
editor@mycotaxon.com

Pacific Northwest Mycology Service
6720 NW Skyline Boulevard
Portland, Oregon 97229-1309 USA

NOMENCLATURE EDITOR

SHAUN R. PENNYCOOK
PennycookS@LandcareResearch.co.nz

Manaaki Whenua Landcare Research
Auckland, New Zealand

MyYcoTAxoONn, LTD. © 2022

www.mycotaxon.com &
www.ingentaconnect.com/content/mtax/mt

P.O. BOX 264, ITHACA, NY 14581-0264, USA

IV ... MYCOTAXON 137(1)

MY COTAXON

VOLUME ONE HUNDRED THIRTY-SEVEN (1) — TABLE OF CONTENTS

ROVIC WEISS 5 sick ite hn ale OE 6 OEP hte note FORE wards ob obi ehete » vi
Nomenclatural novelties Oty pificanens sch bey nin soo ten pie has She vii
COPFICON OG gu arte chert ithe potter ce cap shee te ob cae SEES kg bel ateak ene ee viii
PROMI UOEAIOF va Gi & cooks 32 Soke SARL Bath Bee oR PPE EE cok bos ix
BOD NSO MISSTONEDTOCR AINE: price pes oe sack cee PAC ACR OM GER EL xi

NEW GENERA & SPECIES

Phaeocollybia chefensis sp. nov. and new synonyms for
P rifflipes, P. rufotubulina, and P. tibiikauffmanii
LORELEI L. NORVELL, RONALD L. EXETER, MATTHEW GORDON,
SAHRA- TAYLOR MULLINEUX, SCOTT A. REDHEAD

Beltrania shenzhenica sp. nov. from
Guangdong Province, China ZHAO-XUE ZHANG, TAI-CHANG Mu,
ZHUANG LI, XI1U-GUO ZHANG, JI-WEN XIA
Termitomyces cryptogamus sp. nov.
associated with Macrotermes natalensis in Africa
LENNART J.J. VAN DE PEPPEL, Z. WILHELM DE BEER,
Duur K. AANEN, BEN AUXIER

Clitopiloides prati and Trichopilus lecythiformis spp. nov.
from Australia Davip L. LARGENT & MOLLy B. CRIBARI

Neoacrodictys elegans gen. & sp. nov.
from Hainan Province, China
JI-WEN X14, TaI-CHANG Mu, ZHAO-XUE ZHANG,
ZHUANG LI, XIU-GUO ZHANG

Neotypification of Claviceps humidiphila
and recognition of C. bavariensis sp. nov.
M1ao Liu, Ey1 TANAKA, MIROSLAV KOLARIK

NEW COMBINATION

Passalora golaghati comb. nov. from India
GARGEE SINGH, SANJAY YADAV,
RAGHVENDRA SINGH, SHAMBHU KUMAR

31

4]

51

63

73

89

JANUARY-MARCH 2022... V

NEW RANGES/HOSTS

Stigmatomyces aff. limnophorae
on dipteran hosts in Peninsular Malaysia
NATASHA AZMI Nour ALIAH, JINGYU LIv,
NuRUL AZMIERA, CHONG CHIN HEO 95

Pluteus variabilicolor and Volvopluteus earlei,
new records for Pakistan
JUNAID KHAN, HASSAN SHER, AIMAN IZHAR,
MUHAMMAD HAQNAWAZ, ABDUL NASIR KHALID 109

New Turkish records of Hebeloma excedens and H. geminatum,
and confirmation of H. celatum
AYTEN D1zkIRICI, AYSENUR KALMER, ISMAIL ACAR 123

Ganoderma multipileum and Tomophagus cattienensis—
new records from Pakistan
AISHA UMAR, SHAKIL AHMED,
LauRA GUZMAN-DAVALOS, MILAY CABARROI-HERNANDEZ 135

Marasmius tageticolor and M. tucumanus
from the Dominican Republic
Nico.tAs NIVEIRo, NATALIA A. RAM{REZ, CLAUDIO ANGELINI 153

MycosBiotTa (FUNGA) NEW TO THE MYCOTAXON WEBSITE
New records of Chaetomium and Chaetomium-like species
(Ascomycota, Chaetomiaceae) on Syagrus coronata
from the Raso da Catarina Ecological Station (ESEC),
Caatinga, Bahia, Brazil (SUMMARY)
NILO GABRIEL SOARES FORTES & NADJA SANTOS VITORIA 171

VI ... MYCOTAXON 137(1)

REVIEWERS — VOLUME ONE HUNDRED THIRTY-SEVEN (1)

The Editors express their appreciation to the following individuals who have,

prior to acceptance for publication, reviewed one or more of the papers

prepared for this issue.

Mustafa Emre Akcay
Joe Ammirati

Flavia Rodrigues Barbosa
Timothy J. Baroni

Sarah Bergemann

R.E Castaneda Ruiz
Tobias Guldberg Froslev
Danny Haelewaters
Egon Horak

Alfredo Justo

Abdullah Kaya

Rachel A. Koch

N’golo Abdoulaye Koné
Jian Ma

Li-Guo Ma

Brandon Matheny

Roger Fagner Ribeiro Melo
Nelson Menolli Jr.

Lorelei L. Norvell

Jadson José Souza de Oliveira
Shaun R. Pennycook

Walter P. Pfliegler

Mario Rajchenberg

Scott A. Redhead

Gerardo Robledo

Michelline Lins Silvério
Adna Cristina Barbosa de Sousa

Joey B. Tanney

JANUARY-MARCH 2022... VII

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 137(1)

Beltrania shenzhenica Z.X Zhang, J.W. Xia & X.G. Zhang
[MB 839268], p. 36
Claviceps bavariensis M. Kolarik, E. Tanaka & M. Liu
[MB 838352] p. 80
Claviceps humidiphila Pazoutova & M. Kolarik 2015 (neotypified)
[MBT 395372], p. 78
Clitopiloides prati Largent
[IF 558177], p. 53
Neoacrodictys J.W. Xia & X.G. Zhang
[MB 816515], p. 66
Neoacrodictys elegans J.W. Xia & X.G. Zhang
[MB 816516], p. 66
Passalora golaghati (Saikia & Sarbhoy) Gargee Singh, Sanj. Yadav,
Raghv. Singh & Sh. Kumar
[MB 835579], p. 90

Phaeocollybia chefensis Norvell & Exeter
[IF 559441], p. 12

Termitomyces cryptogamus van de Peppel
[MB 838129], p. 44

Trichopilus lecythiformis Largent
[IF 558178], p. 56

vill ... MYCOTAXON 137(1)

CORRIGENDA

MYCOTAXON 136(4)

p.v,line9 FOR: Five new foliicolous micromycete records from Turkey

READ: Additions to the knowledge of foliicolous micromycetes in Turkey

JANUARY-MARCH 2022... IX

FROM THE EDITOR-IN-CHIEE

LATE AGAIN! — Unseasonal snowfalls, power & computer outages at critical times,
the Covid Omicron variant in New Zealand, time-consuming medical treatments
in Oregon, research obligations, and fewer final submissions (despite a last-minute
25-day extended deadline) have all conspired to delay our 2022 January-March
MycotTaxon. Your editors regret the unavoidable delay and heartily thank all authors
for their extreme patience. The GoopD news is that nomenclatural reviews are now
being returned very quickly by a recovered Nomenclature Editor and soon-to-be
acknowledged final submissions are arriving at an equally brisk pace. Being realistic,
we suspect the April-June issue will probably go out in early (if lucky) July rather
than late June, but your two volunteer editors are valiantly trying to bring back the
journal to the quick turn-around its founders intended. Welcome to the slim—but
mighty—MycotTaxon 137(1).

Our 2022 January-March MycoTaxon may indeed be slender, but it offers a
wide variety of truly fascinating papers. There are 13 contributions by 49 authors
(representing 16 countries) as revised by 28 expert reviewers and the editors.

The NEW GENERA & SPECIES Section proposes ONE new genus (Neoacrodictys from
China) and SEVEN species new to science representing Beltrania & Neoacrodictys
from Cuina; Claviceps from GERMANY; Clitopiloides & Trichopilus from AUSTRALIA;
Phaeocollybia from the U.S.A.; and Termitomyces from SOUTH AFRICA. We also
offer one new combination in Passalora from India, and one newly registered
neotypification for Claviceps humidiphila from its type locality in Japan.

The NEW RANGES/HOSTS section contains five titles. Species range extensions are
reported for [basidiomycetes] Ganoderma & Tomophagus and Pluteus & Volvopluteus
from PaxisTan; Hebeloma from Turkey; Marasmius from the Dominican
Republic; and [laboulbeniomycetes] a possible new Stigmatomyces species from
MataysiaA. New dipteran hosts (Boettcherisca and Hypopygiopsis [the first for any
laboulbenialean species] are cited for Stigmatomyces. MycoTAxON 137(1) also
provides keys to species in Neoacrodictys, Passalora, and Phaeocollybia (the last also
proposing three long-awaited synonymies). And a first: papers providing conclusions
supported by sequence analyses include all seven newly described species (including
one cryptospecies diagnosed by sequence data alone—for good reason) and all eleven
range extensions.

Our issue concludes with the announcement of a new annotated species list on our
MYCOBIOTA website, which describes and illustrates six new records and includes a
key to Chaetomium and Chaetomium-like species in one Caatinga ecological research
station in BRAZIL

Warm regards,

Lorelei L. Norvell (Editor-in-Chief)
20 April 2022

x ... MYCOTAXON 137(1)

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-SIX (4)

MYCOTAXON for OCTOBER-DECEMBER 2021 (I-XLVIII + 693-880)
was issued on February 8, 2022

JANUARY-MARCH 2022... XI

2022 MyCOTAXON SUBMISSION PROCEDURE

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The MycoTaxon journal publishes four quarterly issues per year. Both open access
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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 1-30
https://doi.org/10.5248/137.1

Phaeocollybia chefensis sp. nov. and new synonyms for
P. rifflipes, P. rufotubulina, and P. tibiikauffmanii

LORELEI L. NORVELL’, RONALD L. EXETER?, MATTHEW GORDON’,
SAHRA- TAYLOR MULLINEUX‘, SCOTT A. REDHEAD‘

' Pacific Northwest Mycology Service, 6720 NW Skyline, Portland OR 97229 USA

72477 SW Maplewood Drive, Dallas OR 97338 USA

> Molecular Solutions, 4216 N. Castle Ave., Portland OR 97217 USA

‘ National Mycological Herbarium (DAOM), Agriculture & Agri-Food Canada,
960 Carling Avenue, Ottawa ON KIA 0C6 Canada

*CORRESPONDENCE TO: I/norvell@pnw-ms.com

ABSTRACT—Phylogenetic analyses of ITS and RBP2 sequence data from Phaeocollybia
collections made at Cascade Head Experimental Forest in Oregon support recognition
of a new species, P. chefensis. Collections of the new species were previously referred to
P. tibiikauffmanii. Sequence analyses also establish that P tibiikauffmanii is a synonym
of P. spadicea, P. rifflipes is a synonym of P lilacifolia, and P. rufotubulina is a synonym of
P. californica. A revised general key to Pacific Northwest Phaeocollybia species is provided.

KEY worps—Basidiomycota, Hymenogastraceae, nomenclature, Northwest Forest Plan,
taxonomy

Introduction

Phaeocollybia R. Heim (Agaricomycetes, Hymenogastraceae) is a genus of
brown-spored agarics characterized by conic-campanulate pilei, cartilaginous
stipes and pseudorhizae, ornamented “beaked’ basidiospores, sarcodimitic
tissue, monovelangiocarpy, and tibiiform diverticula that arise from the
mycelium and primordial pellicular remnants (Norvell 1998a,b; Norvell
& Exeter 2009). The forests of western North America have provided an
exceptionally large number of species in this genus (Murrill 1911; Smith 1937,
1957a,b; Smith & Trappe 1972; Redhead & Norvell 1993; Norvell 1998a,b,

2 ... Norvell & al.

2000, 2002, 2004; Norvell & Redhead 2000, Norvell & Exeter 2007, 2009;
Norvell & al. 2010).

In 1995 the first author collected a solitary tawny colored Phaeocollybia
from Oregon's Cascade Head Experimental Forest (CHEF). Although the
specimen’ general morphology resembled that of P kauffmanii (A.H. Sm.)
Singer, it differed by possessing abundant refractive tibiiform cheilocystidia,
prompting application of the provisional name P. tibiikauffmanii to the
Oregon coast taxon in Norvell’s 1998 doctoral dissertation.

The author’s research on Phaeocollybia coincided with establishment
of the Northwest Forest Plan (NWFP; USDA-USDI 1994), which listed 14
phaeocollybias among its 234 fungal species of concern (Castellano & al.
1999, 2003; ORBIC 2021). In 1998 transects were established for surveying
epigeous ectomycorrhizal basidiomycetes in Benton County’s Bureau of
Land Management (BLM) Green Peak Density Management Study and
Polk County’s BLM Fungal Chronosequence Study (Norvell & Exeter 2004).
Numerous gregarious to caespitose clusters of P kauffmanii-like specimens
with tibiiform cheilocystidia were collected that matched the solitary 1995
specimen from Lincoln County. After morphological comparison with
other specimens from Washington, Oregon, and California, a particularly
well-documented collection from the chronosequence ‘old growth’ study
transect (approximately 64 km from the CHEF site) was selected as type for
P. tibiikauffmanii (Norvell 2004).

In 2008, the U.S. Forest Service (FS) & BLM Interagency Special Status/
Sensitive Species Program initiated a project to generate DNA sequence data
for Phaeocollybia specimens (Gordon 2009). Since then, 350 sequences for
the internal transcribed spacer region (ITS1-5.8S rRNA-ITS2) in the nuclear
ribosomal RNA gene cassette and 46 sequences of a portion of the gene
encoding the second largest subunit in the DNA-directed RNA polymerase
(RPB2) were deposited in GenBank. These data, along with NWFP surveys,
have provided several hundred Phaeocollybia collections for taxonomic and
genetic analysis, and serve as an excellent resource for phylogenetic analyses
and specific primer design.

Initial molecular analyses that clustered P. tibiikauffmanii and P. spadicea
sequences within one intermixed clade were received on the same day as
the arrival of the galley proofs for PHAEOCOLLYBIA OF PACIFIC NORTHWEST
NortTH AMERICA (Norvell & Exeter 2009). Although the vital data arrived
too late for a thorough publication revision, the authors annotated their
keys and commentaries in anticipation of subsequent taxonomic changes.

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 3

Additional ITS sequence analyses confirmed the synonymy of P. spadicea and
P. tibiikauffmanii but also supported newly collected material from the original
Lincoln County ‘tibiikauffmanii site as a distinct species, independent of
P. spadicea.

A revised ITS-based phylogeny of Phaeocollybia was presented in the
Norvell & al. (2010) poster at the International Mycological Congress in
Edinburgh. In this paper we provide additional ITS and RPB2 sequence data
that establish P chefensis as a new species and formally publish synonymies
for P. californica (= P. rufotubulina), P lilacifolia (= P. rifflipes), and P. spadicea
(= P. tibiikauffmanii).

Materials & methods

Collections

Specimen collection and examination, ultraviolet inspection, and syringaldazine
spot tests of fresh material followed procedures outlined in Norvell & Exeter (2009),
where morphotaxonomic terms (e.g., tibiiform diverticula, sarcodimitic tissues,
pellicular veil, vertical-monopodial, and rhizomorphic pseudorhizae) are also defined.
Parenthesized color references from Ridgway (1912: e.g., “Pale Pinkish Cinnamon”)
accompany colors of fresh specimens described in general non-standardized color
names in lower case. Microscopical examinations were made of fresh tissues in H,O
or (for the type material) dried tissues rehydrated in 6% aqueous KOH. Basidiospores
were taken from the stipe apex. Dimensions of all anatomical cells follow the format
n (number measured): (outlier)low-average-high(outlier) [e.g., basidia n = 19:
25-34.2-41(43) x 6.5-8.1-9.4(9.9) um].

Collector abbreviations include LLN (Norvell), RLE (Exeter), and sAR (Redhead).
Latitude/longitude geographic coordinates were converted and rounded to four
decimal places from surveyor input using [for TRS] https://www.earthpoint.us/
TownshipsSearchByDescription.aspx and [for UTM] https://www.ngs.noaa.gov/
NCATY/.

Vegetation abbreviations follow those used during Northwest Forest Plan surveys
and include (in all caps for overstory) ABPR (Abies procera Rehder), PISI (Picea
sitchensis (Bong.) Carr.), PSME (Pseudotsuga menziesii (Mirbel) Franco), and TSHE
(Isuga heterophylla (Raf.) Sarg.) and (in small caps for understory) BENE (Berberis
nervosa Pursh = Mahonia nervosa (Pursh) Nutt., the preferred synonym), GASH
(Gaultheria shallon Pursh), oxor (Oxalis oregana Nutt.), Pomu (Polystichum munitum
(Kaulf.) C. Presl), vaov (Vaccinium ovatum Pursh), and vapa (Vaccinium parviflorum
Andrews = Gaylussacia baccata (Wangenh.) K. Koch, the preferred synonym).

Material cited in the SPECIMENS EXAMINED Sections is housed (unless otherwise
specified) in Norvell’s Pacific Northwest Mycology Service fungarium (PNW) and
will be distributed among DAOM, NY, OSC, PNW, and WTU after publication.
Herbarium acronyms follow Thiers (2021).

4 ... Norvell & al.

TABLE 1: PNW Phaeocollybia ITS & RPB2 sequence data. (Type data in bold).

SPECIES

ammiratii

attenuata

benzokauffmanii

californica

#

#

chefensis

dissiliens

fallax

COLL./FUNG.
NUMBER

LLN1941028-10-T
LLN2051018-01
OSC 155802
RLE2007-103
RLE2007-105
RLE2008-068
RLE2009-19
RLE2009-31
RLE2009-41
LLN2071029-51
RLE2007-026
RLE2007-090
RLE2007-176
LLN1921120-01-T
RLE2007-035
RLE2009-15
MICH 11607-T
OSC 109290
OSC 109332
RLE2007-177
RLE2010-05
LLN1921116-1 ruf-T
SAR7500
RLE2009-04-T
LLN2091026cc6a
OSC 155803
RLE2007-074
RLE2007-129
OSC 114217
RLE2008-138
LLN1921007-01
RLE2004-01
RLE2007-140
RLE2007-159
RLE2007-175

ITS

JN102495
GQ165629
KJ450913
(GQ165627)
GQ165624
KX574502
JN102493
JN102494
KX574503
JN102501
MZ352106
JN102498
(GQ165632)
GQ165636
GQ165636
(JN102502)
JN102503
EU669240
EU846292
GQ165647
JN102504
GQ165707
GQ165708
MZ352102
MZ352101
MK326851
MZ352097
MZ352096
EU846271
KE219569
JN102512
JN102511
MZ352103
JN102516
JN102518

RGB2

KU574760
KU574759
KU574787
KU574790

KU574794

KU574761

KU574785

KU574764

KU57484
KU574779
KU574767

KU574791
KU574795
KU574796

CouNTY, STATE/PROV;
PRIMARY COLLECTOR

Clackamas, OR; Norvell
Skamania, WA; Norvell
Coos, OR; Rodenkirk
Benton, OR; Exeter

Polk, OR; Exeter

Benton, OR; Exeter
Benton, OR; Exeter
Benton, OR; Exeter
Benton, OR; Exeter
Benton, OR; Norvell
Lincoln, OR; Exeter
Benton, OR; Exeter
Benton, OR; Exeter
Mendocino, CA; Norvell
Benton, OR; Exeter
Benton, OR; Exeter

Del Norte, CA; Smith
Linn, OR; Smith
Josephine, OR; Friend
Benton, OR; Exeter
Benton, OR; Exeter
Mendocino, CA; Norvell
Mendocino, CA; Redhead
Lincoln, OR; Exeter
Benton, OR; Norvell
Polk, OR; Christensen
Lincoln, OR; Exeter

Lane, OR; Exeter

Coos, OR; Sperling
Benton, OR; Exeter
Vancouver Isl. BC; Redhead
Benton, OR; Exeter
Benton, OR; Exeter
Benton, OR; Exeter

Benton, OR; Exeter

SPECIES

gregaria

kauffmanii

lilacifolia

A

A

luteosquamulosa

ochraceocana

olivacea

oregonensis

COLL./FUNG.
NUMBER

LLN2091026cg8
RLE2007-061
RLE2008-026
RLE2013-07b
LLN1931015-02
RLE2006-10
RLE2006-13
RLE2007-095
RLE2008-027
RLE2008-045
LLN1921111-06
LLN2071018-12
RLE2006-43
RLE2013-44
LLN2071029-33
LLN2071029-43
RLE2007-016
WTU-F-003084
RLE2007-033-T
OSC 134678
OSC 134679
RLE2007-009
LLN1921015-03
LLN1921122-04
LLN1941128-01
OSC 109501
OSC 113875
RLE2007-122
RLE2007-133
RLE2008-050
RLE2008-051
RLE2008-129
LLN2001105-01
OSC 67425
RLE2006-16
RLE2013-42

ITS

JN102520
GQ165652
GQ165654
KJ450915
JN102542
KE219573
JN102547
JN102524
KE219572
JN102526
KE219576
GQ165657
KE219580
GQ165668
GQ165667
GQ165663
JN102528
GQ165674
GQ165672
GQ165671
GQ165673
JN102533
JN102530
JN102532
EU846281
EU846282
JN102534
JN102535
MZ352094
JN102536
GQ165678
GQ165681
EU846273
GQ165685

Phaeocollybia chefensis sp. nov. (U.S.A.) ...

RGB2

KU574772

KU574782
KU574781
KU574763
KU574803
KU574774

KU574775

KU574798
KU574799
KU574766

KU574776

KU574804

CouNTtTY, STATE/PROV;
PRIMARY COLLECTOR

Benton, OR; Norvell
Lincoln, OR; Exeter
Polk, OR; Exeter

Polk, OR; Exeter
Clallam, WA; Leuthy
Benton, OR; Exeter
Benton, OR; Exeter
Polk, OR; Exeter

Polk, OR; Exeter

Lane, OR; Exeter
Clackamas, OR; Norvell
Lincoln, OR; Exeter
Polk, OR; Exeter

Lane, OR; Exeter
Benton, OR; Norvell
Benton, OR; Norvell
Lincoln, OR; Exeter
Snohomish, WA; Ammirati
Polk, OR; Exeter
Benton, OR; Villella
Tillamook, OR; Paque
Polk, OR; Exeter
Jefferson, WA; Norvell
Mendocino, CA; Norvell
Clackamas, OR; Norvell
Coos, OR; Sperling
Douglas, OR; Kersens
Benton, OR; Exeter
Lane, OR; Exeter

Lane, OR; Exeter
Benton, OR; Exeter
Benton, OR; Exeter
Multnomah, OR; Norvell
Douglas, OR; Goldenberg
Lane, OR; Exeter

Lane, OR; Exeter

6 ... Norvell & al.

SPECIES

phaeogaleroides

piceae

pleurocystidiata

pseudofestiva

radicata

redheadii

scatesiae

sipei

spadicea

P sp. 1

COLL./FUNG.
NUMBER

RLE199-064a-T
RLE2009-29a
RLE2010-086
MICH 11629-T
RLE2007-178
RLE2009-12

LLN1940330-02-isoT

LLN1930516-01
RLE2008-002
LLN1921104-10
RLE2007-069
RLE2007-070
OSC 112980
RLE2006-19
RLE2008-131
LLN2071018-17
LLN2091026cg18
RLE2007-106
RLE2008-038
LLN1921015-19
LLN1931104-09
LLN2071029-02
RLE2007-151
LLN1971023-69
OSC 96908
RLE2007-123
OSC 112482
OSC 113791
OSC 134542
RLE2009-18
RLE2009-20

A2011031o0x1 tib-T

A206111402-O
RLE2007-165
OSC 155805
RLE2010-05
RLE2014-01

ITS

OM065392
ME737171
KX574499
ME737169
(MZ352093)
KE219583
GQ165688
GQ165686
GQ165687
KE219588
KE219589
KE219590
EU846275
GQ165696
GQ165695
JN102544
JN102541
JN102546
MZ352098
GQ165701
GQ165703
GQ165699
GQ165700
EU644706
EU644707
GQ165704
EU697252
EU669364
MZ352099
JN102550
JN102551
KF219597
KEF219596
(KE219594)
JN102507
JN102504
KU574726

RGB2

KU57489

KU574762

KU574765

KU574797

KU574771

KU574783

KU574768
KU574769

KU574773

KU574786
KU574788
KU574780
KU574778
KU574777
KU574793

KU574801

County, STATE/PROV;
PRIMARY COLLECTOR

Benton, OR; Exeter
Benton, OR; Exeter
Benton, OR; Exeter
Tillamook, OR; Smith
Mendocino CA; Bojantchev
Benton, OR; Exeter
Clallam WA; Norvell
Linn, OR; Bailey
Benton, OR; Exeter
Multnomah, OR; Norvell
Lincoln, OR; Exeter
Lincoln, OR; Exeter
Coos, OR; Rodenkirk
Benton, OR; Exeter
Benton, OR; Exeter
Lincoln, OR; Norvell
Benton, OR; Norvell
Polk, OR; Exeter
Lincoln, OR; Exeter
Jefferson, WA; McClenaghan
Lincoln, OR; Norvell
Benton, OR; Norvell
Benton, OR; Exeter
Benton, OR; Norvell
Linn, OR; Bacheller
Benton, OR; Exeter
Benton, OR; Giachini
Douglas, OR; Wetzel
Douglas, OR; Sperling
Benton, OR; Exeter
Benton, OR; Exeter
Polk, OR; Norvell
Polk, OR; Norvell
Benton, OR; Exeter
Lane, OR; Loring
Benton, OR; Exeter

Benton, OR; Exeter

Phaeocollybia chefensis sp. nov. (U.S.A.) ...

ey eee COLL./FUNG. ITS RGB2 CounrTY, STATE/PROV;
NUMBER PRIMARY COLLECTOR

P sp. 2 RLE2009-09 KX574498 _ Benton, OR; Exeter
RLE2010-109 KX575400 — Benton, OR; Exeter

P. sp. 3 LLN2071029-53 (KF219598) | KU574770 Benton, OR; Norvell
RLE2007-100 KX574493 aan Polk, OR; Exeter
RLE2007-152 KF219600 _ Benton, OR; Exeter
RLE2015-35 KU574725 KU574800 — Benton, OR; Exeter

P sp. 4 RLE2010-009 MZ352104 KU574792 —_ Benton, OR; Exeter
RLE2015-01 KX574505 _ Benton, OR; Exeter
RLE2015-06 KU574727 KU574802 Benton, OR; Exeter

Psp. 5 OSC 155806 MF957115 = Josephine, OR; Scelza
RLE2013-14 KJ450918 — Benton, OR; Exeter

P. sp. 6 OSC 134575 GQ165677 — Linn, OR; Nakvasil
OSC 151647 MH819350 MH823881 Douglas, OR; Scelza

# = P. rufotubulina, = P rifflipes, and * = tibiikauffmanii in
Norvell (1998ab, 2002, 2004) and Norvell & Exeter (2004, 2007, 2009).
(T = type); ITS sequences enclosed in parentheses not included in Fie. 1.

DNA amplification & sequencing

Standard protocols and published primers were followed for DNA extraction,
PCR amplification, and sequencing (White & al 1990, Gardes & Bruns 1993, Liu &
al. 1999, Matheny 2005). Amplifications of the ITS region from 350 Phaeocollybia
collections representing 34 putative species utilized fungal primers ITS1 and ITS4b
(Gardes & Bruns 1993). Amplifications of the RPB2 gene region from 46 collections
representing 26 putative species utilized the degenerate basidiomycete specific
primers bRPB2-6F and bRPB2-7.1R (Matheny 2005). Additionally, a new RPB2
forward primer, RPB2Phf (5’-GcAGAAACACCYGAGGGC-3’, slightly downstream
from the bRPB2-6F primer) was designed based on existing Phaeocollybia RPB2
sequences and used successfully where the original primer pair failed after multiple
attempts. In total 396 (350 ITS + 46 RPB2) new sequences were generated. Additional
ITS and RPB2 sequences were added from GenBank to provide a comprehensive
phylogeny for Phaeocollybia (Norvell & al. 2010, this publication). TABLE 1 provides
collection and sequence data for PNW specimens, and TABLE 2 provides sequencing
information for extralimital taxa and the outgroup cited in Fics 1 and 2.

Separate gene regions were initially aligned with Clustal X (Thompson &
al. 1997). Subsequent new sequences were aligned manually in MacClade 4.0
(Maddison & Maddison 2000). Taxa for which gene regions were not sequenced
were coded as missing. Sequences were aligned using MAFFT ver. 7 (Katoh & al.
2019) and manually corrected using MEGA X 10.0.0 (Kumar & al. 2018, Stecher &
al. 2020). All new sequences were deposited in GenBank.

8 ... Norvell & al.

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Phaeocollybia chefensis sp. nov. (U.S.A.) ..

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10 ... Norvell & al.

TABLE 2: Sequences from extralimital Phaeocollybia spp. and outgroup
used in phylogenetic analyses

SPECIES FUNGARIUM # GENBANK # COUNTRY
christinae MCVE 3539 JF908573 Italy
cidaris O-F-252891 UDB036543 Norway
elegans PDD 72723 KY827313 New Zealand
festiva (ITS) WTU-F-053245 DQ494682 Norway
— (RPB2) WTU-F-053245 AY509118 Norway
gracilis PDD 88665 KY827314 New Zealand
graveolens PERTH 5311586 AF501567 Australia
lugubris O-F-253794 UDB037370 Norway
MCVE 14619 JF908574 Italy
megalospora SP 445402 KC662116 Brazil
pakistanica-T SWAT 15-1560 KY007615 Pakistan
SWAT 15-1561 KY007616 Pakistan
ratticauda PDD 72678 KY827316 New Zealand
PDD 72544 KY827315 New Zealand
tenuis PDD 72672 KY827318 New Zealand
Phaeocollybia sp. TRTC 157723 BOLD CRA031-09 Costa Rica
AAM A1064 KF041417 Mexico
PDD 71198 KY827317 New Zealand
Galerina WTU-F-007080 DQ486706 Washington USA
semilanceata
(RPB2 gene) WTU-F-007080 AY337357 Washington USA

All sequences represent the ITS region unless otherwise indicated.

Sequence analyses

Sequences were analyzed using programs available in Geneious Prime (version
2021.2.2). Sequence data were obtained from NCBI and compiled into a multiple
sequence alignment. The original ITS dataset contained 296 sequences identified as
Phaeocollybia spp. Duplicate sequences were removed. Representative sequences for
each taxon were retained and the final dataset, including the outgroup, comprised
129 unique sequences. Sequences were trimmed to remove the CATTA and GACCT
motifs. For the analysis of partial RPB2 gene sequences, the dataset was composed of
49 unique sequences.

Sequences were aligned automatically using the Geneious alignment tool, and the
alignment was refined manually. Phylogenetic analyses were carried out using the
MrBayes 3.2.6 (Huelsenbeck & Ronquist 2001) plugin developed in Geneious. The
following parameters were used: substitution model GTR; rate variation gamma;
gamma categories 4; 5 million generations; subsampling frequency 1000; burnin

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 11

Galerina semilanceata PBM 1398 (WTU) (AY337357)

KUS574789 (Oregon) P. phaeogaleroides

P. olivacea

P. gregaria
P. olivacea

Phaeocollybia sp. 6

Avs00118 (Nowy) festival
1.90(KU574782 (Oregon)
1,00{ (LKUS74781 (Oregon)

P. ammiratii

Poregonensis
-P. benzokauffmanii
00} (00 | | KU574778 (Oregon) Pana
KUS574788 (Oregon)
KU574786 (Oregon)
oo(-KU574724 (Oregon)
KUS574761 (Oregon) P. attenuata
"Hg XU574802 (Oregon)
1.01
eee (Oregon) Phaeocollybia sp. 4
5 KU57484 (Oregon)
"i P. chefensis
Y KU574779 (Oregon)

KU574771 (Oregon) P. radicata

Fic. 2. Phylogenetic analysis of the partial RPB2 gene region in strains of Phaeocollybia spp.
Branch lengths were determined using the Bayesian consensus outfile. Nodes with posterior
probability values <0.90 are indicated in blue. Type strains are indicated in bold.

frequency 50%; heated chains 4; heated chain temperature 0.2; unconstrained branch
lengths GammaDir (1, 0.1, 1, 1); and shape parameter exponential (10). The outgroup
was Galerina semilanceata PBM 1398 (WTU); DQ486706 for the ITS analysis and
AY 337357 for the RPB2 analysis, respectively). Phylogenetic trees were drawn using
the Bayesian consensus outfile and annotations were added using Inkscape 0.92.4.

Phylogenetic results FIGs 1-2

Norvell & al. (2010) presented the first comprehensive ITS phylogeny for the
genus based on 300 Phaeocollybia sequences (including two from Australia—
P. graveolens B.J. Rees & K. Syme, P. ratticauda E. Horak—and P. festiva (Fr.)

12 ... Norvell & al.

R. Heim from Norway) and the outgroup—Hebeloma radicosum (Bull.)
Ricken, Psilocybe semilanceata (Fr.) P. Kumm., Galerina marginata (Batsch)
Kuhner—revealing a cohesive clade of 69 putative taxa with the Galerina-
like P phaeogaleroides clade in the basal-most position within Phaeocollybia.
The current ITS tree (pruned to eliminate duplicates) confirms the existence
of three synonymies, several difficult species complexes, and six unnamed
species (Fic. 1). The three synonymies supported are P rifflipes = P. lilacifolia
(>99.7% similarity), P rufotubulina = P. californica (>99.5% similarity), and
P. tibiikauffmanii = P. spadicea (>99.8% similarity (Fics 1, 2).

The comprehensive ITS phylogeny (not shown) generated from 296
sequences (plus the outgroup Inocybe pallidicremea Grund & D.E. Stuntz [as
I. lilacina (Peck) Kauffman)] and Galerina semilanceata (Peck) A.H. Sm. &
Singer) and the trimmed 129-sequence ITS tree (Fic. 1) both support 29 PNW
species and 14 extralimital putative taxa. A familial relationship between
Phaeocollybia and Galerina is also supported, with both genera now classified
in Hymenogastraceae (Matheny & al. 2006, Kalichman et al. 2020).

The successful amplification of far fewer (46) RPB2 sequences prohibits
generating a robust multigene consensus tree. Nevertheless, both ITS (Fic. 1)
and RPB2 (Fic. 2) trees support the same clades. With insufficient coverage
across the genus for 28S sequence data and with the two-gene phylogeny
well supported by convincing morphological characters, we do not include a
28S-based phylogeny at this time.

Taxonomy

Phaeocollybia chefensis Norvell & Exeter, sp. nov. Fics 3-6
IF 559441

Differs from Phaeocollybia kauffmanii by its tibiiform cheilocystidia and from P. spadicea
by its uniformly carrot orange to orange-tawny coloration, larger basidiospores, and
solitary to scattered (never gregarious or cespitose) habit.

TyPeE: Oregon, Lincoln County, Cascade Head Experimental Forest, W of Hwy 12, 30m S
Tillamook Co. line, 45.0447°N 123.9153°W, 245 m asl, 27.x.2009, Tsuga heterophylla (old
and young), Picea sitchensis, Vaccinium parviflorum, Polystichum munitum, RLE2009-04
(holotype OSC 155804, isotype PNW; GenBank MZ352102, KU574784).

EryMo.oey: derived from the acronym (CHEF) for the type locality, Cascade Head
Experimental Forest.
PitEus 70-80 mm diam, broadly conic-campanulate with acute papillate
umbo, upturned inner margin, down-turned outer margin, and straight edge;
glabrous, glutinous in rain with the gluten at times somewhat milky, non-striate,
overall brownish orange or foxy brown (Tawny, Orange Cinnamon); dried

Ron Exeter

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 13

SS
—
|
?
!

Le,

Fic. 3. Phaeocollybia chefensis (Holotype, RLE2009-04). Top: excavated specimens with intact
vertical monopodial pseudorhizae. Bottom: Two views of the type collection in situ in Cascade
Head Experimental Forest, Lincoln County, Oregon. Five collections of 1-2 specimens were
collected from this same site during 1995-2021.

pileus uniformly copper metallic. CONTEXT ~6 mm at the disc and confluent
with stipitipith, pale orangish white. Opor faintly floral with farinaceous
overtones; TASTE mild, not distinctive. LAMELLAE nearly free, ventricose,
thin with + even edges, polydymous with 3-7 irregularly interspersed tiers of
lamellulae, narrow (4-5 mm, with average length/width ratio 5.5), close, pale
orangish buff (Warm Buff) when young, developing darker spots in maturity.
VEIL sparse (when present, evident only as occasional darker fibrillose

14 ... Norvell & al.

remnants on stipe apex). STIPE slightly eccentric, terete, 80-90 mm above
ground level, overall length including pseudorhiza <200 mm, apex 10-12
mm diam, + equal above, gradually narrowing below toward pseudorhiza;
glabrous except for occasional fibrils, moist, finely longitudinally lined, apex
pale to deep pinkish orange (Pinkish Cinnamon, Orange Cinnamon) below
grading to dull pinkish brown (Fawn Color), 2 mm thick cartilaginous rind
surrounding compact fibrillose orangish white stipitipith, rind sometimes
with small perpendicular separations producing ~5 mm long recurved rind
patches. PSEUDORHIZAL FORM vertical-monopodial, <2/3 overall stipe length,
gradually tapering to pale salmon-colored pointed to blunt origin, firm pith
brown where water-soaked, otherwise concolorous with stipitipith. SPORES IN
MASS dull pinkish brown (Fawn Color).

BASIDIOSPORES (n = 64): 8-8.9-10 x (5)5-5.6-5.8 um, Q = (1.45)1.52-
1.65-1.81(1.84), limoniform with a protruding beaked apex in profile,
fusoid-elliptical to amygdaliform in face view, apical callus 0.5-1 um
long (occasionally abrupt or more often tapering to end), ornamentation
verruculose to verrucose except on smooth apical callus and eccentric
apiculus, suprahilar plage an indistinctly bordered area of lowered
ornamentation (oil immersion); orangish amber in KOH (ochraceous in
H,O, based on examinations of paratypes). Basip1A (n = 19): 4-spored,
curved; broadly clavate above narrower base, 25-34.2-41(43) x 6.5-8.1-
9.4(9.9) um, sterigmata 2.0-3.0-3.7 um long, curved; hyaline to dull orangish
brown, guttulate, granular, or uniformly oily. CHEILOCYSTIDIA 24.2-28.7-34
um long, diameters (basal septum) 2-2.6-3 x (stomach) 4.9-5.7-6.9 x (neck)
1-1.3-1.6 x (capitulum) 1.5-2.0-2.6 um (n = 13), abundant, intermixed with
basidia, secretory; capitulate tibiiform (primarily) and lageniform, (only
occasionally intermixed with thin-walled clavate elements), bases hyaline,
necks and capitula refractive, thick-walled, pale amber. PLEUROCYSTIDIA
absent except for isolated cheilocystidia scattered occasionally on the gill face.
LAMELLAR TRAMA hyphae parallel, 65-80 x 3-6 um, thin-walled, hyaline
inflated, subgelatinized, narrowing toward gill edges to 2-3 um diam and
giving rise to the subhymenial layer. SUBHYMENTIUM c. 25 um thick, hyphae
2-4 um diam, tightly packed, parallel, cylindrical, hyaline, gelatinized.
PELLICULAR HYPHAE: PILEIPELLIS a bilaminate ixocutis with a >300 um thick
SUPRAPELLIS with elements tightly compact in freshly dried material (but
readily separating in gel matrix after 11 years in fungarium), hyphae >40 x
1.5-5.0 um, radially aligned, sinuous, long-branching, gelatinized, frequently
(spirally) gel encrusted, hyaline; sUBPELLIS c. 500 um thick, gelatinized, vessel

A-C, E-G: Lorelei Norvell. b, 4,1. Ron Exeter 1: Heidi Christensen.

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 15

Fic. 4. Phaeocollybia chefensis paratypes. [Lincoln Co.: LLN1951109-16 (A-C), RLE2007-074 (F),
RLE2015-21 (H), RLE2021-1 (J); Lane Co.: RLE2007-129 (D, E); Polk Co.: HC2018-LB4 (I); Benton
Co.: LLN2091026cc6 (G)]. A-C. Three views of the first specimen from CHEF that fostered the
concept for P. tibiikauffmanii in 1995. D. Young robust specimens excavated from Lobster Creek.
E. Pristine pseudorhizal tissues display strong magenta reaction in syringaldazine after 15 minutes.
F. Second specimen retrieved from CHEF in 2007. G. Solitary specimen retrieved from Conner’s
Camp on Marys Peak. H. Basidiome at the original CHEF site prior to excavation in 2015. I. The
first primordium excavated with its more mature partner in 2018 at Boulder Creek. J. Exeter’s
2021 negative syringaldazine reaction from this waterlogged solitaire cautions that adverse
environmental conditions do disable this usually reliable diagnostic tool.

16 ... Norvell & al.

hyphae with thinner walls; hyphae long, 4-12 um diam at septa and inflating
to <25 um; pigments orange to dark brownish orange, diffuse to occasionally
encrusting, soluble in KOH (forming droplets in mountant); small hyaline
crystals scattered infrequently throughout. STIPITIPELLIs hyphae 75-100 x
1.5-3(6) um, parallel aligned, gelatinized, pigments diffuse, pale amber to
reddish in places. RHIZOPELLIS cells long x 4-10 um, pale to dark amber,
heavily gel- and/or red-brown pigment-encrusted. TRAMAL TISsUEs: lightly
(pileus) to noticeably (stipe, pseudorhiza) sarcodimitic, gelatinized, +hyaline
except darkening in pseudorhiza; VESSEL HYPHAE fusoid (sometimes tapering
to obtuse end), 25-100 x 3-13 (at septa), rigid walls 1-2 um thick; FLExUOUS
HYPHAE generally winding or curving around vessels, more or less cylindrical
but sometimes appearing flattened, thin-walled, <50 um long, diameters
narrowest (1-2 um) in pileus and broadest (3-5 um) in stipe; OLEIFEROUS
HYPHAE occasional to frequent in tramal tissues throughout, sinuous,
aseptate, 3-10 um diam, thin-walled, contents dull brown, oily. Trs11FoRM
DIVERTICULA infrequent on aerial stipe apex, lageniform to (more often)
tibiiform, frequent to abundant on rhizopellis, 8-20 um long, 1-2 at base,
narrowing in refractive neck area before expanding when capitula (1-2
um diam) present; arising directly from hypha and lacking basal septum,
secretory, hyaline, refractive to very pale amber. CLAMP CONNECTIONS absent
in all tissues.

SYRINGALDAZINE SPOT TEST: strongly positive (pseudorhizal origin dark
burgundy in five minutes; stipe (cross-section) and pileus and lamellae
leaching magenta after ten minutes). FLUORESCENCE fresh material (of
holotype) not tested; dried lamellae dull yellow orange (one small area of one
specimen a brilliant orange yellow).

ADDITIONAL SPECIMENS EXAMINED—UNITED STATES, OREGON, Benton Co.
Conner’s Camp, 44.5060°N 123.5565°W, 762 m asl, 200yo PSME TSHE 26.x.2009
LLN2091026cc6 (MZ352101). Lane Co. Lobster Valley, 44.2268°N, 123.6148°W, 427
m asl, 200yo PSME young TSHE Gasu, 6.xi.2007 RLE2007-129 (MZ352096). Lincoln
Co. Cascade Head EF, 30 m S Tillamook Co. Line, 45.0447°N 123.9153°W, 245 m asl,
~120yo PISI TSHE vapa POMU OxOR: 9.xi.1995 LLN1951109-19; 18.x.2007 RLE2007-074

Fic. 5. Phaeocollybia chefensis (Holotype, RLE2009-04): A. Colorless suprapellis in gelatinous
matrix overlying a pigmented subpellis [400x]. B. Pileipellis under oil [1000x]. C, D. Suprapellis
hyphae (gel encrustations detailed in C). E. Lightly sarcodimitic pileus tramal tissues with
relatively thin-walled vessel hyphae. F. Oleiferous hyphae in stipititrama (just below pellis
hyphae). G. Strongly sarcodimitic pseudorhizal trama with thick-walled vessel hyphae and thin-
walled flexuous (arrow) hyphae.

Fe

Phaeocollybia chefensis sp. nov. (U.S.A.) ...

[[2410N 1a[al0]

18 ... Norvell & al.

(MZ352097, KU574779); 2.xi.2015 RLE2015-21 (KX574495); 14.x.2021 RLE2021-01.

Polk Co. Boulder Creek 44.8983°N 123.4995°W, 823 m asl, ~80yo ABPR PSME TSHE

POMU VAPA 10.x.2018 HChristensen HC2018-LB4 (OSC 155803; MK326851).
ECOLOGY & DISTRIBUTION: autumn (October-November); basidiomes solitary
or in pairs in needle duff under mature Abies procera, Tsuga heterophylla,
Picea sitchensis and/or Pseudotsuga menziesii with understory of Gaultheria
shallon, Oxalis oregana, Polystichum munitum, Vaccinium ovatum, and/or
V. parviflorum. Known thus far from only five sites along the Oregon coast and
in the Oregon coast range.

Discussion

Phaeocollybia chefensis

Diagnostic characters include a bright orange to brownish orange viscid
conic-campanulate pileus, seemingly robust stature that in age becomes limp
or flaccid, stuffed stipe, vertical monopodial pseudorhiza, medium-sized
limoniform verrucose basidiospores, thick-walled tibiiform cheilocystidia, and
lack of clamp connections. ITS and RPB2 sequence analyses strongly support
P. chefensis as an independent species (Fics 1, 2).

Morphologically, all paratypes match the type, except for the smaller (7.5-
8-9 x 4—-4.5-5.2 um) basidiospores measured in the immature 1995 specimen
(LLN1951109-19), which in part explains why Norvell (2004) did not detect
the selection of a P spadicea collection as type until after sequence analyses.
The 1995 collection has not been sequenced, but sequences from the four
subsequent collections from the identical site strongly support the earlier
collection as P. chefensis. The molecularly confirmed collections imply Tsuga
heterophylla as the primary ectomycorrhizal associate; possible secondary
associates include Picea sitchensis at the type locality (245 m asl), Pseudotsuga
menziesii at the Lobster Creek (427 m asl) and Conner’s Camp (762 m asl)
sites, and Abies procera and Pseudotsuga menziesii at Boulder Creek (823 m
asl). Lamellae of fresh specimens examined under UV exhibited the strong
yellow fluorescence characteristic of all PNW phaeocollybias except for the
uniquely non-fluorescing P gregaria A.H. Sm. & Trappe.

As noted above, Phaeocollybia chefensis closely resembles P. kauffmanii
macroscopically, while microscopically it is quite similar to P spadicea and
P. pseudofestiva A.H. Sm., Phaeocollybia chefensis shares with P. kauffmanii a
brownish orange viscid pileus, stuffed stipe, vertical-monopodial pseudorhiza,
magenta syringaldazine reactivity, and morphologically similar basidiospores,
but the presence of refractive thick-walled tibiiform cheilocystidia and

Phaeocollybia chefensis sp. nov. (U.S.A.) ..

“Se. Sr en)

3

~ ee

Lorelei Norvell

Fig. 6 Phaeocollybia chefensis (Holotype, RLE2009-04 (A,D,E); RLE2007-074 (B,C); RLE2007-129
(4,B): A, B. Cheilocystidia. C. Granular basidia, sterigmata, and orangish brown basidiospores
with smooth apical beaks (arrow). D. Basidiospores with older basidia lacking guttules or granules.

E. Comparison of verrucose spore ornamentation and less ornamented plage regions (arrows).
FE Abundant tibiiform diverticula on pseudorhizal pellis.

“

|

kd

20 ... Norvell & al.

the absence of heavily gelatinized, strongly sarcodimitic elements in the
stipe and less extensive gelatinous matrix in the suprapellis clearly separate
P. chefensis from P. kauffmanii. In the field the much longer-lived P kauffmanii
can be distinguished by its larger (at times massive) and more robust stature,
sharp farinaceous odor and taste, strongly inrolled mature pileus edge, and
frequently gregarious habit.

Phaeocollybia spadicea, sharing many morphological features that led to
the selection of an orange form as type for P. tibiikauffmanii, differs in its
darker tawny (“date-colored”) to frequently blackish brown pileus, abundant
rough fibrillose patches (invariably covered with tibiiform diverticula) on
the stipe apex, closely gregarious habit, and (often) negative reactivity of the
pileus and lamellar tissues to syringaldazine. Specimen age and condition
may explain the variable syringaldazine reactivity in both P chefensis and
P. spadicea.

Phaeocollybia pseudofestiva, which shares general basidiospore and
cheilocystidial morphology, syringaldazine reactivity, and odor and taste
is easily separated from P. chefensis by its dark green pileus, smaller size,
more prominently beaked basidiospores, cord-like pseudorhiza, and closely
gregarious habit.

Phaeocollybia tibiikauffmanii and P. spadicea

As noted above, comparisons of ITS and RPB2 sequences from collections
designated as either P spadicea or P. tibiikauffmanii based on the presence/
absence of orange coloration (Norvell 1998a,b,c, 2004; Norvell & Exeter 2009)
revealed that all (including the holotype) except one (Fic. 3a) P. tibiikauffmanii
collection represented P spadicea. Subsequent morphological comparisons
have shown that P. spadicea pileus colors range along a foxy orange to tawny to
deep brown continuum. Additionally, all seven P. chefensis collections occurred
singly or in pairs in contrast to P. spadicea, which typically occurs in closely
gregarious to cespitose clusters; Alexander Smith's notes archived in MICH
describe an “orange” spadicea that he provisionally named P. “caespitosa”
(a name he also provisionally applied to what was to become P. scatesiae A.H.
Sm. & Trappe; Norvell 1995). Thus, we propose the synonymy:

Phaeocollybia spadicea A.H. Sm., Brittonia 9: 215, 1957. FIG. 5
= Phaeocollybia tibiikauffmanii Norvell, Mycotaxon 90: 248, 2004.
SPECIMENS CONFIRMED AS Phaeocollybia spadicea (listing those previously cited as
P. tibiikauffmanii in Norvell (2004) and/or Norvell & Exeter (2004, 2009)—UNITED
STATES. OREGON: Benton Co. Conner’s Camp 44.5060°N 123.5565°W, 762 m asl,

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 21

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CG Elixabeth Fox.

Fic. 7. Phaeocollybia spadicea [RLE208-55 (A); A201103102-1 (B); LLNI1921015-10 (c)].
A. Gregarious cluster on Oregon’s Marys Peak (as P. tibiikauffmanii in Norvell & Exeter
2009). B. Excavated P. tibiikauffmanii holotype from the Polk County Chronosequence Study.
C. “Typical” brown-capped P. spadicea specimens in Hoh Valley, Olympic National Park.

200yo PSME TSHE, 5.xi.2005 RLE2008-55 (KF219592); Ernest Creek 44.4185°N
123.5165°W, 610m asl, 200yo PSME TSHE 15.xi.2007 RLE2007-165 (KU574777,
KF219594); Running Bear 44.4774°N 123.5769°W, 696 m asl, 60yo PSME TSHE,
6.xi.2000 DCalver RLE200-103 (KF219595). Polk Co. Pedee Chronosequence Study
44.8113°N 123.5212°W, 460 m asl, 150yo PSME TSHE pomu 4.xi.1998 (all by LLN
& RLE) A198110401-01, A198110402-02-04 (with CHibbler); 5.x.2000 RLE200-
045; 18.x.2000 A200101801-15,02-25; 1.xi.2000 RLE200-091, A200110102-30;
31.xi.2001 A2011031lox1 (P_ tibiikauffmanii holotype; KF219597, KU574780),
A201103102-03,04,05,06,08, A2011031ox-02; 14.xi.2011 «a201111401-01,02,03,
A20111140x-01; 15.x.2002 =a202101502-01; = 13.xi.2002 = =a202111302-01,02,03;
14.xi.2006 A206111402-O (KF219596, KU574778), RLE2006-47, 16.x.2007 rle2007-
060 (KF219593). WASHINGTON: Jefferson Co. Hoh Valley @ Twin Creek 47.8307°N
124.0012°W, 180 m asl, ~400yo PISI TSHE pomMu oxor 15.x.1992 LLN1921015-10 .
(w sAR) (WTU-F-003698; previously cited and RFLP-confirmed as P. spadicea).

dsl

ADDITIONAL EXAMINATION NEEDED BUT PROBABLY ALSO Phaeocollybia P. -UNITED
STATES. OREGON: Benton Co. Green Peak BLM Density Management Study 44.366°N
123.455°W, (all Gp collections by LLN & RLE): Clear-cut transect (pre-treatment)—
610m asl, 65yo PSME TSHE pomu 24.xi.1998 GP1981124c1-04 (w TFennell); High
Retention transect 579m asl, 65yo PSME TSHE pomu— 8.xii.1999 GP1991208hx.03
(w sAR) (PNW, DAOM), 27.xi.2000 GP2001127hx3; Klickitat BLM Unit-3 44.4406°N
123.5673°W, 60yo PSME TSHE GasH 30.x.2000 RLE200-065; 16.xi.2000 RLE200-252;
Klickitat Unit-6 T.S. 44.4548°N 123.5473°W, 50yo PSME TSHE pomu: 30.x.2000
RLE200-082,83,90, 14.xi.2000 RLE200-187b; 44.4260°N 123.5472°W, 60yo PSME
TSHE GasuH: 30.x.2000 RLE200-070; RUNNING BEAR TS. 44.4774°N 123.5770°W,
200yo PSME TSHE pomu 9.xi.1998 LLN1981109.108e w RLE, 6.xi.2000 RLE200-103;
28.x.2002 RLE2002-16. Linn Co. Keel Flats T.S. 44.5133°N 122.6518°W, PSME BENE
POMU 6.xii.1999 KScott RLE199-BK. Polk Co. Pedee Crk head waters near Cold Springs
44.8233°N 123.4862°W, 610m asl, 200yo PSME TSHE Gasu 26.xi.2001 RLE 2001-
112,115. WASHINGTON: Clallam Co. Olympic NF Klahanie Campground 47.9657°N
124.3057°W, 250yo PISI TSHE 18.x.1992 STrudell LLN1921018-4 __, (WTU-F-003646).

usps3

22 ... Norvell & al.

Phaeocollybia rufotubulina and P. californica

On November 16, 1992, numerous densely gregarious orange
phaeocollybias in Jackson State Forest (Mendocino County, California) were
collected and referred to P californica. This first highly informative collection
contained numerous pin-head primordia that helped establish the presence
of a universal veil for all Phaeocollybia species and introduced the ‘sequential-
racemose’ pseudorhiza arising from a horizontal ‘mother rhizomorph within
the soil (Norvell 1998b, 2004).

Restriction Fragment Length Polymorphism (RFLP) analyses of the ITS
region from type materials supported separation of P rufotubulina from
P. californica and P. scatesiae (Norvell 1998a), indicating a new species and
supporting the synonymy of P. scatesiae under P. californica proposed by Horak
(1977). The 2009 ITS sequence analyses, however, clustered P californica and
P. rufotubulina within one intermixed clade well-separated from P. scatesiae,
a brownish species macroscopically easily distinguishable in the field from
the two orange phaeocollybias. Phaeocollybia scatesiae is now supported as
independent from P. californica (Fics 1,2).

Given the difficulty encountered in isolating DNA from the P. californica
holotype (obtained in 2011), it is probable that the 58-year-old P californica
DNA isolated in 1994 was contaminated by DNA isolated in the same run
from the much younger P scatesiae isotype. We hereby propose the synonymy:

Phaeocollybia californica A.H. Sm., Brittonia 9: 216, 1957.
= Phaeocollybia rufotubulina Norvell, Mycotaxon 90: 243, 2004.

Norvell & Exeter (2009) presented P. californica (pp. 63-70) and P. rufotubulina
(pp. 175-180) as separate species; all material related to P rufotubulina
throughout that monograph as well as those in Norvell (1998a,b, 2004) should
now be referred to P. californica.

Phaeocollybia rifflipes and P. lilacifolia

During a two-month long collecting expedition through western North
American coastal rainforests 1992, several small lilac-gilled tawny to dark
brown-capped phaeocollybias were collected and provisionally determined
as P. lilacifolia. Except for flaccid small basidiomes and unusually small
basidiospores, the specimens morphologically matched Smith's P. lilacifolia
type from Washington’s Mt. Rainier Park (Smith 1957a). However, 1994
restriction digests produced a RFLP profile unique to these collections.
Unable to extract DNA from the Washington P /ilacifolia holotype or Smith's

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 23

Cascade Head Experimental Forest paratype, Norvell (1998a, 2002) proposed
the name P rifflipes (honoring its unique RFLP fingerprint) for the specimens
that were morphologically separated by unusually small basidiospores and
stature.

During 2001-2013, numerous small P. rifflipes specimens were collected
from the Polk County old growth chronosequence transect; not until 2007,
however, was the first recognizable P. lilacifolia collected from Lincoln
County's Saddle Bag Mountain, c. 25 km southeast of Cascade Head
Experimental Forest. The Saddle Bag collections comprised robust healthy
specimens with normal-sized basidiospores. In 2009, ITS phylogenetic
analyses clustered P lilacifolia sequences and all but one P rifflipes sequences
within one clade, a clade supported by our current ITS and RPB2 phylogenies.
Disposition of 4592sl (MZ352100), a Loring collection from Josephine
County (initially identified as P. rifflipes), remains unresolved at this time.

Although we have not yet successfully isolated DNA from Smith's
P. lilacifolia holotype or paratypes, the small “limp” stature, poor condition,
and age of the 1992-1994 collections suggest that their peculiar RFLP
profiles resulted from an unknown contaminant. The following synonymy
is proposed:

Phaeocollybia lilacifolia A.H. Sm., Sydowia Beih. 1: 59, 1957.
= Phaeocollybia rifflipes Norvell, Mycotaxon 81: 102, 2002.

All keys, descriptions, and photos of P. rifflipes in Norvell (1998a, 2002) and
Norvell & Exeter (2009) should be referred to P. lilacifolia.

Revised general key to Pacific Northwest Phaeocollybia species
Basidiospore size ranges of measurements taken from holotype specimens are

cited for species; means of measurements taken from molecularly confirmed
specimens are cited for complexes.

1. Basidiomes small: pileus <50 mm diam, and never green or drab;

stipe apex usually <4 mm diam and never drab....................2200005 2
1. Basidiomes medium to large: pileus usually [50 mm diam;

stipeapex tsttally. SSimummsdiamy 4h. -aaths.- 2aihaegdige+adthar 2 4thernw A Qaenuld haceuls 5

2. Basidiospores ellipsoid, ornamentation minutely punctate and
small apical callus detectable in 1000x oil immersion;
Clainp-CcONMECtONS PrESENL, 52. os see < +a als alka sew polled ON gle OR Ga OE 3
2. Basidiospores limoniform, ornamentation verrucose and
protruding apical beak visible without oil immersion lens;
ClaniIp,COnNeEChOnis-ADSCM ts ys, Beet Re rc ty Raney Muay Bendy Menten tenet cathy wate 4

24 ... Norvell & al.

3. Spores small (5-6 x 3-3.5 um); cheilocystidia lageni-/tibiiform,

necks narrow and thick-walled, abundant but inconspicuous,

difficult to see in dense gelatinous matrix;

basidiome-collybicid, stipeplaable-. cis T icons rs 20h. ype ne ape ere eee P. radicata
3. Spores larger (~10 x 6 tum); cheilocystidia thin walled,

narrowly clavate, extending well beyond hymenium;

basidiome mycenoid, stipe fragile and easily broken . . . P. phaeogaleroides complex

4. Stipe and pseudorhiza fleshy, not shiny or brittle;

cheilocystidia tibiiform, necks and capitula refractive and thick-walled;

pleurocystidia present and, frequent on gill faces;

spores 8-11 x 5-7 um, tapering to straight beaks;

Phenology: Veriiah F.-:o0 vos yaa oa: Monies Nowute Homie Howse He P. pleurocystidiata
4. Stipe polished, corneous, soon hollow;

pseudorhiza lateral monopodial, brittle and wire-like (criniform);

cheilocystidia clavate, thin-walled; pleurocystidia absent;

spores 7-8.5 x 5-5.5 um, big-bellied with abrupt tilted beaks;

prenologscatiturinmale st cit tn shot hin tg hle ty phat oiahed Cee P. attenuata
5. Young pileus green, rapidly aging to brown or brownish olive .................. 6
5. Young pileus ochre, orange, tawny, brownish, or drab (never green) ............. 8

ON

. Young lamellae violet; spores ~9 x 5.3 um, moderately beaked,

in face view fusoid or naviculate, verrucose to marbled;

cheilocystidia clavate, thin-walled, apices swollen to subcapitate,

only rarely forming filiform apical outgrowths .............. P. fallax complex
6. Young lamellae creamy to yellowish buff; spores limoniform with
pronounced beak, +/- ovate in face view, rugulose to warty roughened;
chéilocystidiasclavate or Tibiitoritit. 4.2.0.3 ela ete lends sate ee alas vi

7. Spores 7.5-8 x 4.5-5 um, with long, projecting beaks;

cheilocystidia tibiiform with narrow refractive necks ......... P. pseudofestiva
7. Spores ~10 x 6 um, ‘turtle-backed’ with abruptly protruding

eccentric refractive beaks; cheilocystidia thin-walled, clavate,

filiform apical outgrowths frequent in age ................ P. olivacea complex

8. Spores ellipsoid, short (<7.5 um), punctate-roughened;

cheiloeystidid-clavate, thin-walled: . ota. ete sla poled a tends atta sigan ntar sian 9
8. Spores limoniform, verruculose to warty;

cheilocystidia tibittorimiorelavater oe. a6. le donated set ae karst sleas ecigs 11

9. Basidiome drab to gray, robust; stipe <20 mm diam, stout, firmly stuffed;
pseudorhiza fleshy; taste and odor cucumber-farinaceous;
spores 6-7.5 x 4-4.5 um; all tissues soon deep magenta in
SVE AMAZING: \ 0M et esc eee hee Ng EE Teh Ae Ue et Ee P. oregonensis
9. Basidiome orange to auburn colored, fragile; stipe <13 mm diam,
slender, hollow; pseudorhiza cord-like; taste and odor mild;
pilets:cclamellacsyringaldazine negative c.% ae aid Sh We ls hon wheal ances vices 10

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 25

10. Clamp connections abundant; pileus orange with yellowish margin,
viscid, conic-campanulate; cheilocystidia filamentous to narrowly clavate;
SPORES515= O15 9CB, GS SMe fast yd pestis ¢ otis se wha siiee etal iay tor alats ee BS P. dissiliens
10. Clamp connections absent; pileus uniformly auburn colored,
subviscid, campanulate with a raised blunt umbo;
cheilocystidia narrowly clavate with long pedicels and subcapitate apices;
SPOTLESS. Or oN aa aa ha 8a a tal ae P. sipei

1

—

. Pileipellis 3-layered with colorless gelatinized middle layer

between yellow to yellow-orange top and bottom layers;

pileus minutely scaly (appressed), dry to subviscid (never glutinous) ........ 12
1

_

. Pileipellis 2-layered, with colorless top layer and variously pigmented lower layer;
pileus bald, smooth (never appressed scaly), subviscid to glutinous ......... 13

12. Spores large, 9-11 x 5-6.8 um;

pileus dry to greasy, yellowish ochre to ochraceous gold;

frequently associated with Abies ................. 0.0 eee P. luteosquamulosa
12. Spores smaller, 7.2-8.7 x 4.4-5.5 um;

pileus greasy to subviscid, tawny ochraceous or tawny;

associated with Pseudotsuga or Tsugd ........ cece eee eee P. ochraceocana

13. Cheilocystidia tibiiform, broad bases thin-walled,

capitula and narrow necks refractive and thick-walled;

Stipeliollowrors tuted hs oso s.iha 290s ge spares Sasa nt Crh eee nN altel 14
13. Cheilocystidia variably cylindrical to clavate,

filamentous apical secondary growth occasional in older specimens

but always lacking refractive thick-walled necks;

stipes. stubbed th: frm Pithy 5.2 eaee eae a Pee Pays eae Coe Pale es Le. ae 17

14. Mature stipe tubular and hollow; basidiomes closely gregarious to fasciculate,

arising from a branching rhizomorphic cord ............. 00. e cece eee eee ies
14. Mature stipe stuffed with firm compact pith; solitary to closely gregarious,

arising from unbranched fleshy (not cord-like) pseudorhiza ................. 16
15. Pileus heavily glutinous, acutely conic-campanulate, yellowish to dark brown;

habit in dense fasciculate mounds; suprapellis thick, colorless,

hyphae kinked, not spirally pigment-encrusted and with septa obscured

by thick gel matrix; spores 8.5-9.5 x 4.5-5.5 UM ...... eee eee eee ee P. scatesiae
15. Pileus moist to viscid, obtusely convex-campanulate, red orange to tawny;

habit in troops, arcs, or (rarely) fasciculate mounds; suprapellis compact,

amber to orange, hyphae spirally pigment-encrusted with refractive septa;

SPOTeS:8-a=1 OMG tiie |. hese he OP RN ose tas ree hy P. californica

16. Pileus foxy orange to orangish brown; stipe apex orange cinnamon colored;
habit solitary or paired, never gregarious; all tissues soon magenta in
syringaldazine; spores 8-10 x 5-5.8 UM... 6. eee eee eee eee P. chefensis
16. Pileus tawny to dark brown; stipe apex pallid to drab;
habit gregarious to fasciculate; only pseudorhiza magenta in
syringaldazine; spores 7.5-9 X 4.5-5 UM ....... 0 ce eee eee eee P. spadicea

26 ... Norvell & al.

17. Clamp connections frequent throughout, most easily seen

in pileus suprapellis and on cheilocystidia;

pileus campanulate with often papillate umbo, tawny ochraceous to tawny;
cheilocystidia cylindrical to narrowly clavate;

S/SLOS REIN: Mz laay Wie Ge eae BIEN 101 Mix i ie PO MOE Py fe Pi ee io ek Pio tt P. ammiratii

17. Clamp connections lacking (rarely in stipitipellis);

18

—_—

9

—

9

pileus shape & color, cheilocystidial shape, and spore size varied ........... 18

. Young lamellae whitish, smoky gray, or deep violet;

young pileus colors tawny, brownish-pink or drab;
pileus and lamellae magenta in syringaldazine .......................004, 19

. Young lamellae pinkish, orangish, or yellowish;

young pileus colors rarely pink or drab;
syrnealdavineweactivityevatted «o,os dase dada tes deca dandea geld cd pondis 4 ehahdio ee 20

. Spores 7.5-9.4 x 4.5-5.5 um; young lamellae intensely bluish lilac to violet;

pileus tawny to deep brown; taste not distinctive;
psetidorhiza syringaldazine tegative::. oiwios es of sont nea cte non ft nen P. lilacifolia

. Spores 9 X 5.5 um;

young lamellae white (pinkish) or ash gray, never lilac or violet;
pileus pinkish, drab, or purplish brown; taste “bitter-cucumber’ farinaceous;
all tissues soon deep magenta in syringaldazine ........... P. benzokauffmanii

20. Young lamellae never fluorescing under UV;

pileus yellow tan, soon darker (cocoa brown); young stipe ivory tan,

lower stipe staining orange or orange banded at ground level;

habit densely gregarious; syringaldazine negative;

Spores Da A SSG MNT ein 2 New 23 ahs inge Sis inge 8 elena Nein eS Sts P. gregaria

20. Young lamellae fluorescing under UV;

21

21

pileus orange or tawny; young stipe apex buff, orange, or pale cinnamon,
lower stipe colors similar to apex, habit scattered to gregarious,
syringaldazein negative or positive; spores 8-12 umlong ................. 21

. Basidiome small to moderately large; stipe apex <12 mm diam (slender),

stipe stuffed but larval infested, leaving a hollow rind at ground level;

pileus conic umbonate, subviscid, uniformly bright apricot-/peach-colored;

all tissues negative in syringaldazine; pileipellis hyphae colorless,

spirally gel-incrusted, subpellis pigments KOH soluble, diffuse;

spores 8-9.5 x 5-6 um, beaks short (<5 pm), straight ............... P. piceae

. Basidiome large to massive; stipe apex 10-25 mm diam (robust),

stipe long-lived, stuffed with firm, insect-free pith;

pileus campanulate with obtuse umbo, viscid to glutinous, orange to tawny;

all tissues strongly magenta in syringaldazine;

pileus subpellis pigment-encrusted, pigments not KOH soluble;

spores with slightly tilted long (<1.5 um) beaks ....................0.0005. 22

Phaeocollybia chefensis sp. nov. (U.S.A.) ... 27

22. Spores 7.5-10 x 4-6 um, limoniform, beak forward-tilted;
pileus +/- uniformly orange, tawny, or orangish brown,
typically drying with a burgundy-colored peaked umbo;
lower stipe and pith staining first orange, then orangish brown;
cheilocystidia cylindrical or narrowly to broadly clavate,
rarely pedicellateand ‘subicapitate i. etch tind ees eee oe P. kauffmanii

22. Spores 8.5-12 x 5-7 um, amygdaliform with long ‘belly; beak back-tilted;
young pileus with reddish tawny umbo, amber margin, and tan edge,
eventually uniformly mahogany or dark reddish brown;
lower stipe and pith staining reddish brown;
cheilocystidia clavate to subcapitate with subglobose apices,
pedivellate;-catermulate, Sune ews et. eis ects weed ores P. redheadii

Research on Pacific Northwest phaeocollybias is ongoing; a fully revised second
edition of PHAEOCOLLYBIA OF PACIFIC NORTHWEST NORTH AMERICA will be
released after several additional taxa have been published.

Acknowledgments

The Pacific Northwest Phaeocollybia consortium wishes to acknowledge earlier
assistance given by individual collectors, government surveyors and agencies,
fungarium curators (BPI, DAOM, F, HSC, MICH, O, OSC, NY, SFSU, TENN, UBC,
UC, WTU), technicians, and others cited in full on p. 205 of Norvell & Exeter (2009).
We also acknowledge the excellent molecular work by M.N. Louise Lefebvre in 2010
at Agriculture & Agri-Food Canada in Ottawa. Kelli Van Norman (Interagency
Special Status/Sensitive Species Program of the Forest Service (Region 6) PNW
Region and Oregon/Washington Bureau of Land Management) is thanked for
facilitating molecular sampling of government Phaeocollybia collections; particular
thanks are due Darci Rivers-Pankratz (also of ISSSP) for providing data on specimens
inventoried for the Northwest Forest Plan, facilitating microscopical reevaluation,
and forwarding Heidi Christensen’s Boulder Creek collection for examination.
We also thank Dr. Joe Ammirati (University of Washington, Seattle, USA),
Dr. Egon Horak (Innsbruck, Austria), and Dr. P. Brandon Matheny (University
of Tennessee, Knoxville, USA) for their greatly appreciated expert presubmission
reviews. Egon Horak is further thanked for sharing his SEM-based basidiospore
measurements for selected type specimens.

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MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 31-39
https://doi.org/10.5248/137.31

Beltrania shenzhenica sp. nov. from Guangdong, China

ZHAO-XUE ZHANG, TAI-CHANG Mu, ZHUANG LI,
X1u-Guo ZHANG, JI- WEN XIA*

Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests,
College of Plant Protection, Shandong Agricultural University,
Taian, Shandong 271018, China

“CORRESPONDENCE TO: xiajiwen1@126.com; xiajw@sdau.edu.cn

ABSTRACT— During a survey of saprophytic microfungi on dead leaves from Futian Mangrove
Nature Reserve in Guangdong Province, China, a new species of asexual ascomycota,
Beltrania shenzhenica, was identified based on morphology and phylogenetic analyses of
partial gene sequences of ITS and LSU. The detailed morphological description, phylogenetic
tree, and photographs are provided.

Key worps—Amphisphaeriales, anamorphic fungi, Beltraniaceae, phylogeny, taxonomy,

Introduction

Beltrania Penz., typified by Beltrania rhombica Penz. (Penzig 1882), is
currently classified in Beltraniaceae, Amphisphaeriales (Wijayawardene &
al. 2020, Zheng & al. 2020). The genus was mainly characterized by mostly
unbranched dark setae with radially lobed basal cells, macronematous
conidiophores arising from basal cells of setae or from separate radially lobed
basal cells, and polyblastic, integrated, terminal, sympodial and denticulate
conidiogenous cells that produce solitary acropleurogenous swollen
separating cells and biconic, spicate or apiculate conidia with a hyaline
equatorial band (Ellis 1971, Seifert & al. 2011, Lin & al. 2017, Hyde & al.
2020). Beltrania contains twenty species, of which eight are represented by
molecular data (Harkness 1884, Wakefield 1931, Hughes 1951, Pirozynski &
Patil 1970, Matsushima 1975, Rao & Varghese 1978, Zhang & Zhang 2003,

32 ... Zhang & al.

Crous & al. 2014, Tibpromma & al. 2018, Bandgar & Patil 2019, Hyde & al.
2020, Zheng & al 2020).

Futian Mangrove Nature Reserve, located northeast of Shenzhen Bay, was
officially created in 1984 and designated as a national nature reserve in 1988.
It belongs to the East Asian monsoon region with humid subtropical climate.
The annual average temperature of 22.4 °C and average annual rainfall of
1700-1900 mm are conducive to the development of various microbial
species (Meng & al. 2013). During our ongoing survey of anamorphic fungi
associated with mangroves in China, we collected a new species representing
Beltrania in the reserve.

Materials & methods

Isolation & morphological analysis

Specimens of dead leaves were collected from Futian Mangrove Nature Reserve,
Shenzhen, Guangdong Province, China, and returned to the laboratory in plastic
bags. The samples were incubated in plastic boxes lined with moistened tissue paper at
room temperature for one week. Tissue pieces (5 x 5 mm) were randomly taken from
the leaf and surface-sterilized by consecutively immersing in 75% ethanol solution
for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing three times in
sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and
then placed on potato dextrose agar (PDA) (Cai & al. 2009). All the PDA plates were
incubated at 25 °C for 2-4 d, and subcultures from the colony margins were inoculated

TABLE 1. Details of strains and sequences of Beltrania and related genera included in
the phylogenetic analyses. New sequences are in bold; ex-(epi)type isolates are
marked with ".

GENBANK ACCESSION NUMBERS

SPECIES VOUCHER
ITS LSU
Beltrania dushanensis GZCC18-0020 MN252875 MN252882
Beltrania krabiensis MFLUCC 16-0257" MH275048 MH260280
Beltrania pseudorhombica CPC 23656! KJ869158 KJ869215
Beltrania querna CBS 126097 MH864016 MH875474
CBS 122.51 MH856775 MH868293
Beltrania rhombica CPC 27482 KX519515 KX519521
CBS 123.58 MH857718 MH869260
strain 10353 — AB496423
CBS 121.50 _ MH868082
Beltrania shenzhenica SAUCC 00617 MW784619 MW784621
SAUCC 0065 MW784620 MW784622

SPECIES

Beltrania sinensis

Beltraniella acaciae
Beltraniella botryospora

Beltraniella brevis

Beltraniella carolinensis
Beltraniella endiandrae

Beltraniella fertilis

Beltraniella humicola
Beltraniella pandanicola

Beltraniella portoricensis

Beltraniella pseudoportoricensis
Beltraniella ramosiphora
Beltraniella thailandica

Beltraniopsis longiconidiophora
Beltraniopsis neolitseae
Beltraniopsis sp.

Castanediella couratarii

Hemibeltrania cinnamomi

Porobeltraniella porosa

Pseudobeltrania cedrelae

Pseudobeltrania ocoteae

Subramaniomyces fusisaprophyticus

Subsessila turbinata

VOUCHER

JS43

JS42

JS260

JS101

CPC 294987

TUFC 100837
GZCC 18-0081
GZCC 18-0082
IFO 9502

CPC 221937
MFLUCC 17-2137
MFLUCC 17-2138
CBS 203.64
MFLUCC 18-0121"
NECCI 3993

CBS 856.70

CBS 145547
MFLUCC 17-2582"
MFLUCC 16-03777
MFLUCC 17-2139?
MFLUCC 17-2140
CPC 221687

TUFC 10081

CBS 579.71

NECCI 3695
NECCI 3997
MFLUCC 17-2141
NECCI 3994
NECCI 3995
NECCI 3996
COAD 2098'

PF9

CPC 262197

CBS 418.95
MFLUCC 15-0831

Beltrania shenzhenica sp. nov. (China) ... 33

GENBANK ACCESSION NUMBERS

ITS
MNO077366
MNO077365
MNO077364
MN077363
KY173389
MN252877
MN252876
KJ869128
MF580247
MF580248
MH858416
MH275049
KX519516
MH859981
MK876377
MG717500
MH275050
MF580249
MF580250
KJ869126
MH860269
KT119564
KX519517
MF580251
KX519518
KX519519
KX519520
MG559548
MG559552
KT950856
EU040241
KX762288

LSU
MNO077266
MNO077265
MN077264
MNO077263
KY173483
AB496426
MN252884
MN252883
DQ810233
KJ869185
MF580254
MF580255
MH870044
MH260281
KX519522
MH871777
MK876416
MG717502
MH260282
MF580256
MF580257
KJ869183
AB496424
MH872031
KT119565
KX519523
MF580258
KX519524
KX519525
KX519526
MG559558
MG559562
KT950870
EU040241
KX762289

34 ... Zhang & al.

onto new PDA plates. Colonies at 7 d and 15 d were photographed using a PowerShot
G7X mark II digital camera. Fungal micromorphological structures were observed
and photographed using Olympus SZX10 stereomicroscope and Olympus BX53
microscope, both fitted with Olympus DP80 high definition colour digital cameras.
All fungal strains were stored at 4 °C in 10% sterilized glycerin for further studies. The
specimens are deposited in the Herbarium of Plant Pathology, Shandong Agricultural
University, Taian, Shandong, China (HSAUP). Ex-type cultures are deposited in
the Shandong Agricultural University Culture Collection, Taian, Shandong, China
(SAUCC).

DNA extraction, PCR amplification, sequencing

Genomic DNA was extracted from colonies grown on PDA based on a modified
CTAB method (Doyle & Doyle 1990). The nuclear ribosomal internal transcribed
spacer (ITS) was amplified and sequenced using the primer pair ITS4/ITS5 (White
& al. 1990), and the large subunit ribosomal RNA gene (LSU) using the primer pair
LROR/LRS (Vilgalys & Hester 1990, Glass & Donaldson 1995).

PCR was performed using an Eppendorf Mastercycler Thermocycler. The DNA
was amplified in 25 uL reaction volumes containing 12.5 uL Green Taq Mix, 1 uL of
each forward and reverse Biosune primer (10 uM), and 1 uL template genomic DNA
in amplifier, and adjusted with distilled deionized water to a total 25 uL volume. PCR
parameters followed were 95 °C for 5 min, followed by 35 cycles of denaturation at
95 °C for 30 s, annealing at 55 °C for 30 s, and a 1-min extension at 72 °C and ending
with a final 10-min elongation at 72 °C. PCR products were estimated visually by
staining with GelRed after 1% agarose gel electrophoresis. Sequencing was performed
bi-directionally by BioSune Co. Ltd. (Shanghai). Consensus sequences were obtained
using MEGA v. 7 (Kumar & al. 2016). All sequences generated in this study were
deposited in GenBank (TABLE 1).

Sequence alignment & phylogenetic analysis

The quality of our amplified nucleotide sequences was checked and combined
by MEGA v. 7 (Kumar & al. 2016), and reference sequences were retrieved from the
National Center for Biotechnology Information (NCBI). Sequences were aligned
using MAFFT v. 7.310 (Katoh & al. 2019) and manually corrected using MEGA
Wi, 2

The combined gene regions were phylogenetically analyzed using Maximum-
Likelihood (ML) and Bayesian Inference (BI). RaxML analyses (using RaxML v.
8.2.9) and Bayesian analyses (using MrBayes v. 3.2.6) were run on the CIPRES
Science Gateway Portal (Miller & al. 2012). Evolutionary models were calculated
using MrModeltest v. 2.3 (Nylander 2004) selecting the best-fit model for each data
partition according to the Akaike criterion. For ML analyses the default parameters
were used and bootstrap support (BS) was carried out using the rapid bootstrapping
algorithm with the automatic halt option. Bayesian analyses included two parallel
runs of 5,000,000 generations with the stop rule option and a sampling frequency
set to each 1000 generations. The 50% majority rule consensus trees and posterior

Beltrania shenzhenica sp. nov. (China) ... 35

Beltrania sinensis Js1 01
“Beltrania sinensis JS42_

74 “ Beltrania sinensis JS260. 4
Beltrania querna CBS 126097

~’ Beltrania querna CBS - |

Beltrania sinensis JS43° 7

SAUCC0065
‘SAUCCo061 | « Bel rania :
Beltrania rhombica CPC 27482 peltrania
Beltrania rhombica CBS 121. 50
Beltrania rhombica strain 10353
y, Pseudobeltrania cedrela COAD
Pseudobeltranie cedrelae PFO
Beltrania rhombica CBS 123 58
seeltrania dushanensis Gzcct 8-0020
Beltrania krabiensis MFLUCC C 16-(
Beltrania pseudorhombica CP

Beltraniella botryospo raT
Beltraniella endiandrae CPC 22

= Beltraniella brevis GZ oa
Beltraniella brevis GZ

Beltraniella ramosiphora MFLUCC
Beltraniella thailandica MFLUCC 16-02
Beltraniella pseudoportoricensis CBS 14554

Beltraniella humicola CBS 203.64

Tm opeltraniella carolinensis |FO 9502
Beltraniella portoricensis CBS 856.70
Beltraniella portoricensis NFCCI 3993
Beltraniella pandanicola MFLUGC
62. ‘,,Beltraniella fertilis MFLUCC 17-2137
Beltraniella fertilis MFLUCC 17-21 38
Beltraniella acaciae CPC 29498
Forobeltraniella porosa Wen
orobeltraniella porosa NFCCI 3995
e20.93t Porobeltraniella porosa NFCCI 996
3X , eZ Pseudobeltrania ocote
oat Hemibeltrania cinnamomit Feta

| "Femibeltrania cinnamomi_
82/0.08 Hemibeltrania cinnamomi MF
Subsessila turbinata MFLUCC 15-(
Beltraniopsis longiconidiophora MFLUCC
Beltraniopsis pete er aItranionaie

‘T ,Peltraniopsis sp. TUFC 10081
te ae = Beltraniopsis neolitseae CPC 221 68
Subramaniomyces fusisaprophyticus ¢ CBS.
Castanediella couratarii CBS 579.71 “Ei

Fic. 1. Phylogram of Beltrania and related genera, generated from Bayesian analysis based on
combined ITS and LSU sequence genes. Bootstrap support values are shown as ML >50% first,
followed by BI >0.90. Some branches were shortened to fit the page - these are indicated by two
diagonal lines and a correction factor indicating the full length. Ex-type/ex-epitype strains are in
bold. Newly generated sequences are indicated in red.

probability (PP) values were calculated after discarding the first 25% of the samples
as burn-in. The resulting trees were plotted using FigTree v. 1.4.2 (http://tree.bio.

36 ... Zhang & al.

ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS v. 5. The individual
gene datasets were assessed for incongruence before being concatenated by
checking their individual phylogenies for conflicts between clades with significant
ML and BI support (Mason-Gamer & Kellogg 1996, Wiens 1998).

Phylogenetic results

The dataset comprised 46 sequences representing 29 species including
Castanediella couratarii (CBS 579.71) as the outgroup. The final alignment
comprised a total of 1549 characters of the combined ITS and LSU including
gaps, ITS: 1-940 and LSU: 941-1549. Of these characters, 1256 were constant,
152 parsimony-uninformative and 141 parsimony-informative. For the BI
and ML analyses, the substitution model GTR+I+G for ITS and LSU were
selected and incorporated into the analyses. The topology of Bayes tree
confirmed the tree topologies obtained from the ML analyses, and therefore,
only the Bayes tree is presented (Fic. 1). In this tree, our two sequences
formed a distinct clade. Therefore, we determined that our strains belonged
to a novel species of Beltrania.

Taxonomy

Beltrania shenzhenica Z.X Zhang, J.W. Xia & X.G. Zhang, sp. nov. Fic. 2
MB 839268

Differs from Beltrania querna by its wider conidia and from B. rhombica by its longer
conidia.

Type: China, Guangdong Province, Shenzhen, Futian Mangrove Nature Reserve, on
dead leaves of a broadleaf tree, 14 July 2020, Z.X Zhang (Holotype, HSAUP 0061; ex-
type living culture SAUCC 0061; GenBank MW784619, MW784621).

EryMo_oey: in reference to the city where the type was found.

CoLonies on PDA at 25 °C in darkness, increasing in diameter by 11-15
mm/d, surface greyish white to black, flat, dense, reverse black to dark black.
Mycelium partly superficial and partly immersed. Stroma absent. Setae
erect, brown, thick-walled, septate, straight to flexuous, conical at the apex,
slightly swollen at basal cell. Conidiophores 58-88 x 3-5 um, aggregated
in dense fascicles, pale brown, cylindrical, septate, unbranched, straight to
variously curved, proliferating sympodially at apex. Conidiogenous cells
terminal, integrated, subhyaline, smooth, holoblastic, polyblastic, with
several flat tipped denticles. Separating cells 8-16 x 5-7 um, subhyaline,
finely roughened, with several apical, flat-tipped denticles. Conidia 28-
33 x 8.5-12 um (including apical appendage), biconic, aseptate, solitary,
acropleurogenous, subhyaline to pale brown, with distinct granules,

Beltrania shenzhenica sp. nov. (China) ... 37

Fic. 2. Beltrania shenzhenica (holotype, HSAUP 0061). a, b. Surface and reverse of colony on
PDA; c. Mycelium on PDA; d. Setae; e, f. Conidiophores, separating cells, conidiogenous cells, and
conidia; g—i. Separating cells; j-1. Conidia. Scale bars: d-] = 10 um.

without median transverse band, apical appendage 5-9 um long, tapering
to an acutely rounded tip, smooth, without a mucilaginous sheath.
ADDITIONAL SPECIMEN EXAMINED: CHINA, GUANGDONG PROVINCE, Shenzhen,
Futian Mangrove Nature Reserve, on dead leaves of a broadleaf tree, 14 July 2020, Z.X.
Zhang (HSAUP 0065; living culture SAUCC 0065; GenBank MW784620, MW784622).
ComMENTs - Based on phylogenetic analysis, our Beltrania shenzhenica
sequences grouped together with B. querna and B. rhombica, but they formed
a distinct clade. Morphologically, B. shenzhenica is most similar to B. querna

38 ... Zhang & al.

and B. rhombica in conidial shape, but B. querna differs by its narrower conidia
(6-8 um wide; Harkness 1884), and B. rhombica differs by its shorter conidia
(25-26 um long; Penzig 1882).

Acknowledgments

The authors express gratitude to Dr. Jian Ma (College of Agronomy, Jiangxi
Agricultural University, Nanchang, China) and Dr. Li-Guo Ma (Institute of Plant
Protection, Shandong Academy of Agricultural Sciences, Jinan, China) for serving
as pre-submission reviewers and to Dr. Shaun Pennycook for nomenclatural review
and Dr. Lorelei L. Norvell for editorial review. This work was jointly supported by
the National Natural Science Foundation of China (Nos. 31900014, U2002203,
31750001) and National Science and Technology Fundamental Resources
Investigation Program of China (2019FY100700).

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MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 41-50
https://doi.org/10.5248/137.41

Termitomyces cryptogamus sp. nov.
associated with Macrotermes natalensis in Africa

LENNART J.J. VAN DE PEPPEL’, Z. WILHELM DE BEER’,
DuurR K. AANEN*, BEN AUXIER’

‘Laboratory of Genetics, Wageningen University,
6700 AA, Wageningen, the Netherlands

*Department of Biochemistry, Genetics and Microbiology,
Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria,
Pretoria, South Africa

CORRESPONDENCE TO: duur.aanen@wutr.nl

ABSTRACT—A new species of Termitomyces symbiotic with the termite Macrotermes
natalensis is described from Africa. As there are no records of field collected basidiocarps
within this lineage, traditional basidiocarp-based morphological taxonomy is not practical.
While basidiocarps may be obtained rarely from incubation of fungal comb fragments, their
practical use for taxonomical purposes is limited. Therefore, the species is described based
on an ITS nucleotide sequence, with comparisons to an asexual culture. Based on samples
with similar ITS sequences, this species is likely associated with multiple termite hosts across
a large part of Africa.

Key worps—Agaricales, Lyophyllaceae, phylogeny

Introduction

In Africa and Asia, a subfamily of the Termitidae, the Macrotermitinae, live
in obligate symbiosis with members of the basidiomycete genus Termitomyces
(Lyophyllaceae). The fungus resides inside the termite nest in so-called
“fungus gardens,’ with mushroom production being periodically triggered
by rain or the potential health of the nest (Koné & al. 2011). The mushrooms
vary in size and are generally medium to large, although some species, such
as T: microcarpus (Berk. & Broome) R. Heim, produce hundreds of small

42 ... van de Peppel & al.

mushrooms. Traditionally, taxonomy in this genus has been based on
basidiocarp morphology, with molecular evidence only recently added
(Freslev 2003, Mossebo & al. 2017).

During research of termites and their associated fungi in Africa,
one lineage was found that associates with Macrotermes bellicosus and
M. subhyalinus when found in northern Africa, but only with M. natalensis
when found in South Africa (Nobre & al. 2011). Surprisingly, sampling
over ten years has not resulted in the discovery of mature basidiocarps of
this species from any of its termite hosts, frustrating efforts for naming this
species. Rarely, while excavating termite mounds, mushroom primordia
are recovered (de Fine Licht & al. 2005; Vreeburg & al. 2020) that can
occasionally be incubated under laboratory conditions in the absence
of termite workers. Presumably due to this laboratory incubation the
mushrooms are misshapen, as their appearance is not consistent with other
known Termitomyces species. This may be similar to development that
occurs in the production of “Enoki” mushroom from Flammulina velutipes
which causes long thin stipes, smaller caps, and pale coloration (Kites &
Navarro-Gonzalez 2015). Despite the need to provide a name for this well-
studied fungus, the lack of basidiocarps has prevented comparisons with
published literature descriptions (Botha & Eicker 1991b), leading to an
argument for using readily obtained asexual cultures as a stable type against
which to compare (Makonde & al. 2013).

As extensive comparisons of ITS sequences from herbarium material
from South Africa recovered no matches with our M. natalensis symbiont
(van de Peppel & al., unpublished data), we provide a description based
on sequence identity and asexual morphology. While there is a previous
description of asexual characteristics from South African Termitomyces,
only mounds producing basidiocarps were used, with M. natalensis mounds
apparently not sampled (Botha & Eicker 1991b). As basidiocarp records
are lacking, asexual cultures are a logical solution to this taxonomic issue.
Three previous publications addressed differences between species in
asexual cultures, and while interspecific differences were found, asexual
characteristics alone were not considered sufficient to delineate species
(Botha & Eicker 1991a,b; Tibuhwa 2012).

Recently an ecological study of Macrotermes symbionts in Kenya
uncovered results consistent with previous sampling of this lineage (Vesala &
al. 2017). Vesala & al. recovered the same fungal species as the one collected
from South Africa (Nobre & al. 2011, de Fine Licht & al. 2005; Vreeburg & al.

Termitomyces cryptogamus sp. nov. (South Africa) ... 43

2020) from mounds of M. bellicosus, M. herus, M. jeanneli, M. michaelseni,
and M. subhyalinus. Vesala & al. (2020), who selected related ITS sequences
from GenBank including symbionts of M. natalensis and M. bellicosus,
found that these formed a monophyletic clade. These sequences form two
well-supported groups with 97% and 98.5% support, with the exception
of a single sequence (GenBank AF357024) isolated from an unidentified
Macrotermes species. Based on these results we describe the species based
on a fungal isolate from South Africa symbiotic with M. natalensis for which
the fungal genome has been published (Poulsen & al. 2014).

Materials & methods

Samples and Isolates

A heterokaryotic culture, Mn103, was obtained by opening a termite mound and
carefully removing nodules containing asexual spores from the fungal combs without
soil contamination. These nodules were placed on agar plates without antibiotics to
establish hyphal cultures. A homokaryotic culture, P5, was obtained by protoplasting
the Mn103 heterokaryotic culture. Additional details regarding sampling, isolation,
and subsequent protoplasting of this culture are found in Poulsen & al. (2014) and
Nobre & al. (2014). A dried holotype specimen of the Mn103 heterokaryotic culture
and a living ex-holotype culture of the P5 homokaryotic culture are conserved at the
Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands (CBS).

DNA extraction, PCR amplification, sequencing

Genomic DNA was extracted from the protoplasted P5 homokaryotic culture using
the cetyltrimethylammonium bromide (CTAB) protocol using mycelium and spores
scraped from a petri dish. The nuclear ribosomal region containing the ITS1 + 5.88 +
ITS2 region (ITS) was amplified using a standard PCR reaction using Promega GoTaq
polymerase and the fungal specific primer ITS1F and the general reverse primer ITS4
(White & al. 1990, Gardes & Bruns 1993). The 28S region (LSU) was amplified using
primers LROR and LR5 (Vilgalys & Hester 1990).

As no basidiocarps have been reported from M. natalensis termite mounds, we also
surveyed herbarium samples from the South African National Collection of Fungi
(PREM) and Schweickerdt Herbarium (PRUM) (van de Peppel & al. unpublished
data).

Sequence alignment & phylogenetic reconstruction

We used a previously published set of ITS sequences (Vesala & al. 2017), six
GenBank sequences from Termitomyces symbiotic with M. natalensis, and a sequence
generated from the specimen we designate here as the type for the new species.
Sequences were aligned using the web software MAFFT v. 7 with default settings
(Katoh & Standley 2013). Maximum likelihood trees were reconstructed using
IQ-TREE v. 2.0.6 with default settings (Trifinopoulos & al. 2016).

44 ... van de Peppel & al.

Culture & microscopy

Cultures were maintained on MYA (20g Malt Extract, 2g Yeast Extract, 1 L H,O),
and incubated at 25 °C. Microscopical examinations were conducted using a Zeiss
Axio Imager Al with 63X objective lens under DIC optics.

Taxonomy

Termitomyces cryptogamus van de Peppel, sp. nov. Fic. 1
MB 838129

Differs from Termitomyces schimperi by its clearly separated LSU sequence; there are no
useful diagnostic differences in the morphology of the asexual morphs of these species.

Type—South Africa, Pretoria, Rietfontein 321-Jr, 25.7292°S 28.2347°E, May 2011,
DK Aanen, heterokaryotic isolate (Mn103) obtained from asexual nodules on a fungal
comb from a mound of Macrotermes natalensis (Holotype, CBS H-24752 [metabolically
inactive dried culture]; living ex-type culture CBS 147190].

ETYMOLOGY: cryptogamus, referring to the hidden marriages of a genetically well-mixed
species without recorded basidiocarps in vivo.
SEXUAL STATE—not observed in vivo.

ASEXUAL STATE—Growth of colonies on MYA medium reaching 5-6 cm
diam. in 3 weeks at 25 °C (somewhat faster at 30 °C). Growth consisting of
white hyphae mostly submerged in agar, with 1-2 mm diam. clusters of asexual
spores produced on the agar surface. Conidia highly variable in size (10-100
mm long) and shape, with 2-5 nuclei per spore. Heterokaryotic colonies
consistently producing heterokaryotic conidia.

ComMMENTS—In the absence of in vivo basidiocarps, T. cryptogamus cannot
be distinguished readily from closely related Termitomyces species based on
asexual characters. However, ITS and LSU sequence analyses clearly separate
a well-supported clade that includes the holotype of T. cryptogamus and
several other strains obtained from fungal combs of Macrotermes natalensis
in South Africa, as well as from those of M. bellicosus, M. herus, M. jeanneli,
M. michaelseni, and M. subhyalinus in Cameroon, Kenya, Senegal, and Ivory
Coast.

Phylogenetic results

NCBI GenBank accession numbers were obtained for the nucleotide
sequences generated from the protoplasted homokaryotic culture P5:
ITS (MW251838), LSU (MW567773), whole genome (GenBank id:
GCA_001263195).

The ITS sequence of our isolate P5 places Termitomyces cryptogamus inside
Group 1 of Vesala & al. (2017). This group includes fungal individuals symbiotic

Termitomyces cryptogamus sp. nov. (South Africa) ... 45

Fic. 1. Termitomyces cryptogamus: morphology. A. Growth on Malt Yeast Agar showing abundant
nodule formation of asexual conidia; B. Close up view of A; C-H. Variable morphology of conidia;
I, J. Morphology of mushrooms on fragments of excavated fungus garden comb produced after
incubation for 5-10 days; note the small caps (<1 cm), which produce viable basidiospores.

with Macrotermes natalensis, M. bellicosus, M. subhyalinus, M. michaelseni,
M. herus and M. jeanneli (Fic. 2). Except for M. natalensis, these termite species
are found with sister species also closely related to Termitomyces cryptogamus.

To compare with other common Macrotermes mound symbionts, we also
extracted DNA from samples of Termitomyces schimperi (Pat.) R. Heim.
However, repeated PCR amplifications of the ITS region were not successful.

46 ...

van de Peppel & al.

DQ436938 Termitomyces sp. M. natalensis South Africa
DQ436958 Termitomyces sp. M. natalensis South Africa
DQ436940 Termitomyces sp. M. natalensis South Africa

MwW251 T. mus M. natalensi: h Afri
DQ494698 Termitomyces sp. M. natalensis South Africa
DQ436957 Termitomyces sp. M. natalensis South Africa
MG283259 Termitomyces sp. M. natalensis

AY764149 Termitomyces sp. M. natalensis South Africa
DQ436956 Termitomyces sp. M. natalensis South Africa
DQ436964 Termitomyces sp. M. natalensis South Africa
GQ383682 Termitomyces sp. M. michaelseni Kenya
GQ383679 Termitomyces sp. M. jeanneli Kenya
GQ383685 Termitomyces sp. M. michaelseni Kenya

AY764150 Termitomyces sp. M. natalensis South Africa
GQ383683 Termitomyces sp. M. herus Kenya
HQ902219 Termitomyces sp. M. bellicosus \vory Coast
HQ902232 Termitomyces sp. M. bellicosus
GQ383687 Termitomyces sp. M. michaelseni Kenya
HQ902224 Termitomyces sp. M. bellicosus
HQ902218 Termitomyces sp. M. bellicosus Cameroon
AF357024 Termitomyces sp.
GQ922682 Termitomyces sp. M. bellicosus Senegal
HQ902227 Termitomyces sp. M. subhyalinus
JF302815 Termitomyces sp. M. bellicosus lvory Coast
HQ902230 Termitomyces sp. M. bellicosus
HQ902229 Termitomyces sp. M. bellicosus
GQ383686 Termitomyces sp. M. michaelseni Kenya
gg ©Q922681 Termitomyces sp. M. bellicosus Senegal
KY197689 Termitomyces sp. M. subhyalinus Kenya
GQ922687 Termitomyces sp. M. nobilis Gabon
9g ©Q922688 Termitomyces sp. M. muelleri Gabon

AF321368 Termitomyces sp. M. muelleri Gabon
GU001666 Termitomyces sp. Macrotermes sp. Malaysia
$f GU001670 Termitomyces sp. Macrotermes sp. Malaysia
AB051889 Termitomyces sp. M. carbonarius Malaysia

AF321362 Termitomyces sp. M. subhyalinus Senegal
HQ902240 Termitomyces sp. M. subhyalinus Benin
GQ922686 Termitomyces sp. M. subhyalinus Senegal
JF302816 Termitomyces sp. M. subhyalinus \vory Coast

EU816416 Termitomyces sp. M. subhyalinus Burkina Faso
KY197700 Termitomyces sp. M. subhyalinus Kenya
JQ088160 Termitomyces sp. Macrotermes sp. Kenya
GQ383676 Termitomyces sp. M. michaelseni Kenya

GQ383677 Termitomyces sp. M. jeanneli Kenya
82 JQ088143 Termitomyces sp. Macrotermes sp. Kenya
GQ383678 Termitomyces sp. M. michaelseni Kenya
pAF321371 Termitomyces sp. M. bellicosus Senegal

85 GQ383675 Termitomyces sp. M. bellicosus Kenya
[--——— EF091678 Termitomyces sp. Macrotermes sp. Thailand
EU816418 Termitomyces sp. M. carbonarius Vietnam

0.05

*

Termitomyces
cryptogamus

Group 1 sensu Vesala & al.

Group 3 sensu Vesala & al.
Group 4 sensu Vesala & al.
Group 2 sensu Vesala & al.

Group 5 sensu Vesala & al.

Group 6 sensu Vesala & al.

Group 7 sensu Vesala & al.

Group 9 sensu Vesala & al.
Group 8 sensu Vesala & al.

2017

2017
2017
2017

2017

2017

2017

2017
2017

Fic. 2. Maximum likelihood tree based on ITS1-5.8S-ITS2 sequences of Termitomyces
cryptogamus and allied Macrotermes symbionts with their host termite species (in bold) and
their collection location. Outgroups were removed to increase readability of the tree. Numbers

at the nodes indicate ultrafast bootstrap values, only significant node values >95% are displayed.

Species delimitation groups with 97% ITS identity (sensu Vesala & al. 2017) are displayed on the

right side of the tree. A sequence of the T. cryptogamus ex-type P5 is underlined and indicated

by an asterisk.

We were able to amplify LSU sequences from both T. cryptogamus and
herbarium samples of T’ schimperi (PREM41964), which were then analysed
together with other GenBank LSU sequences of T’ schimperi and unidentified
M. natalensis symbionts (Fic. 3). The LSU sequence of our P5 isolate was
identical to that from a fungal symbiont of a M. natalensis mound. LSU
sequences from the commonly recovered Macrotermes symbiont, T. schimperi,

were not monophyletic.

Termitomyces cryptogamus sp. nov. (South Africa) ... 47

T. schimperi RSK186 KU933614

T. schimperi tgf18 AY232712

T. cryptogamus P5 MW567773 W/V. natalensis *

83

Termitomyces sp. ZA164 DQ110875 M. natalensis

Termitomyces sp. EF 667085 MW. natalensis

T. schimperi DM24E KY809228

T. schimperi MW567772 M. subhyalinus

100

T. schimperi PREM41964 MW567771

Termitomyces sp. AB073526 Odontotermes sp.

0.006

Fic. 3. Phylogenetic tree based on LSU sequences for Termitomyces cryptogamus. and publicly
available LSU sequences of T’ schimperi, acommon Macrotermes symbiont. Sequence of AB073526
used as an outgroup to root the tree. Node values indicate bootstrap support.

Microscopical investigations revealed highly variable morphology of
conidia harvested from laboratory grown cultures (Fic. 1c-H), which limits
morphological comparison with asexual cultures of other Termitomyces species.

Discussion

There is accumulating evidence, both direct and indirect, that sexual
reproduction does occur in T: cryptogamus. First, sexual reproduction
between strains associated with M. natalensis was inferred as occurring

48 ... van de Peppel & al.

sufficiently frequently (at least 100 sexual events per generation) to explain
the observed signature of free recombination (de Fine Licht & al. 2006).
Second, mating tests between homokaryons retrieved from heterokaryons
demonstrated that the M. natalensis-associated strains represent a single
biological species (Nobre & al. 2014). Finally, strains associated with
M. natalensis were observed to produce mushrooms and viable basidiospores
in vitro (de Fine Licht & al. 2005; Vreeburg & al. 2020). These findings make it
all the more surprising that mushrooms have not been found in nature. One
hypothesis is that sexual reproduction of this species occurs belowground
synchronously with alate dispersal.

Nevertheless, recovery of in-vitro basidiocarps only after prolonged
laboratory incubation prevents their use as a morphological type specimen,
as presumably incubation conditions greatly influence the resulting
morphology. Additionally, the infrequency and unpredictability of these
mushroom primordia in nests precludes their use for identification. Further,
the asexual spores produced are highly polymorphic, and thus cannot be used
for reliable identification with this group. As such, although comparisons
of asexual cultures remain valuable, molecular markers provide the only
reliable way to identify samples (Licking & al. 2020). Based on the work of
Makonde & al. 2013, it is likely that other Termitomyces species exist where
the only practical identification markers will be molecular.

Using the 3% ITS similarity threshold, we find one lineage (group 1,
sensu Vesala & al. 2017) that is distributed across Africa, with hosts differing
geographically, but always within Macrotermes. The different termite
symbionts combined with geographic isolation may indicate barriers to gene
flow. Further study should show whether or not geographically separated
populations of T: cryptogamus, including populations associated with
Macrotermes species other than M. natalensis, all form a single biological
species.

Although comparisons of ITS sequences from T. cryptogamus and
T. schimperi are not currently available, we feel these sequences represent at
least two species for two reasons. Firstly, the fact that we are unable to amplify
the ITS regions successfully using the ITS1F and ITS4 primers, while we can
amplify the LSU sequence indicates that there are likely mutations in the
primer binding site not found in T! cryptogamus (for which ITS sequences
are readily amplified). This suggests that T. schimperi likely has fixed
substitutions not shared with T. cryptogamus. Secondly, the relationships
between the LSU sequences generated from T: schimperi and T: cryptogamus

Termitomyces cryptogamus sp. nov. (South Africa) ... 49

(Fic. 3) show significant genetic difference, although the backbone nodes of
the phylogeny do not receive statistical support of bootstrap values greater
than 70. Additionally, the recovery of two clades of T’ schimperi indicates that
T: schimperi is potentially paraphyletic and deserves further study.

Acknowledgments

The authors thank Tobias Guldberg Froslev (Geogenetics, University of
Copenhagen, Denmark) and N’golo Abdoulaye Koné (Department of Natural
Sciences, Université Nangui Abrogoua, Abidjan, Cote d'Ivoire) for presubmission
review. D.K.A., L.J.J.v.d.P., and B.A. were supported by the Netherlands Organization
for Scientific Research (D.K.A., L.J.J.v.d.P. by VICIINWO 86514007; D.K.A. and B.A.
by ALWGR.2017.010).

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ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 51-61
https://doi.org/10.5248/137.51

Clitopiloides prati and Trichopilus lecythiformis spp. nov.
from Australia

Davip L. LARGENT & MOLLy B. CRIBARI

Biological Sciences, Humboldt State University,
I Harpst St, Arcata CA 95521, USA

“CORRESPONDENCE TO: mrp@humboldtl.com

ABSTRACT—Clitopiloides prati (from northeastern Queensland) and Trichopilus lecythiformis
(from coastal New South Wales) are described as new entolomatoid species based on
morphological characters. Morphological similarities are discussed for these and other
closely related species. We also provide nuclear ribosomal RNA repeat (nrITS) and large
subunit (nrLSU) sequences where obtained

Key words—Agaricales, Basidiomycota, Entolomataceae, taxonomy

Introduction

Agarics are readily recognized as representing Entolomataceae (Agaricales)
by their flesh colored basidiospores that are angular at least in end view; the
family has a cosmopolitan distribution and iscommon in temperate and tropical
ecosystems. Within the family, basidiomes exhibit highly diverse morphologies
and ecologies. One consequence of this diversity is the acceptance of 1848
species as valid by Kalichman & al. (2020), the second highest of all families
classified in the Agaricales.

The multigeneric classification of Entolomataceae utilized in this paper
follows Largent (1994). The reasons for adopting this approach are detailed in
Largent (2020), but important points are reiterated here: a) the basionym for
nearly every genus in Largent (1994) is identical to the infrageneric basionyms
in Romagnesi & Gilles (1979), Noordeloos (1992, 2004), and Noordeloos &
Gates (2012); (b) it is relatively easy to distinguish the different genera visually

52 ... Largent & Cribari

using this classification in the field; c) although a comprehensive classification
based on a phylogenetic and morphological approach in Entolomataceae is not
yet universally accepted, many segregate entolomatoid genera are supported by
phylogenetic analysis (Karstedt & al. 2019, He & al. 2019).

Australian field investigations from 2009-12 within northeastern
Queensland’s Wet Tropics Bioregion and from 2010-12 in the temperate
rainforests of central New South Wales have uncovered several novel species
in the Entolomataceae (Largent & Abell-Davis 2011; Largent & al. 2011a,b,
2013a,b, 2014, 2016; Bergemann & al. 2013). Here we describe and illustrate
two unusual new species from Australia: Clitopiloides prati and Trichopilus
lecythiformis.

Materials & methods

Macro- & micromorphology

Colors were identified subjectively and coded according to Kornerup & Wanscher
(1978) with color plates noted in parentheses (E63) in the species description.
Common or general names for colors used in this paper are found in the Colour
Sample to Colour Name section in the back of the color handbook where each page
presents tables flagging technical names for color by * and placing common names in
italics; we designate the technical color names using quotation marks (e.g., “Saruk’).

Fresh collections were heat dried. Dried basidiomata were sectioned and
rehydrated in 3% KOH. A Nikon Eclipse Ci compound microscope with Lumera
Infinity 2 imaging software was used to examine and measure microscopic features
following Largent (1994). In the taxonomic descriptions x = mean dimensions,
Q = range of length/width ratios from individual structures, Q. = mean of all
individual length/width ratios, and x/y = the total number of structures measured (x)
and the total number of collections examined (y). Specimens were deposited in the
Plant Pathology Herbarium, Orange Agricultural Institute, Orange, New South Wales,
Australia (DAR); or the Fungarium, Biology Department, Humboldt State University,
Arcata, Calfornia, USA (HSC).

DNA sequences

Dried basidiomata were pulverized and DNA extracted according to Largent
& al. (2011a,b). The ITS region (ITS1, 5.88 and ITS2) of the nuclear ribosomal
RNA repeat (nrITS) was amplified with primers ITSIF (Gardes & Bruns 1993)
and ITS4 (White Q al. 1990) and the nuclear large subunit (nrLSU) was amplified
with primers ctb6 and twl3 (White & al. 1990). PCR reactions of the nrITS and
nrLSU were performed in 50 uL reactions following the procedure as outlined in
Largent & al. (2011b) and PCR cycling followed the protocol detailed in Bergemann
& Garbelotto (2006). PCR products were cleaned using 1 uL of ExoSAP-IT (GE
Healthcare) and incubated at 37 °C for 15 min followed by 80 °C for 45 min.
Cycle sequencing was performed at MCLAB (South San Francisco CA) using an

Clitopiloides & Trichopilus spp. nov. (Australia) ... 53

ABI3730XL sequencer to obtain bi-directional sequences. Contigs were assembled
and edited with Sequencher 4.8.

Taxonomy

Clitopiloides prati Largent, sp. nov. FIG. 1
IF 558177

Differs from Entoloma cyathus by its greyish brown, appressed fibrillose pileus,
equal stipe, encrusting pigments, pileipellis with erect to suberect distal cells, longer
cheilocystidia, ammonia-like odor, and habitat in a lawn.

Type: Australia, Queensland, Cook Region, Smithfield, 10 Marcia Close, 16.8261°S
145.6857°E, gregarious in lawn, 27 September 2010, Peter Newling 40a (Holotype HSC
A1429); GenBank M“W520128, MW520127)

EryMoLoGcy—the genitive of the Latin partum, referring to the habitat in a lawn.

BASIDIOMATA Clitocyboid. PrLEus 30-70 mm broad, <25 mm high, plano-
convex to plane, deeply depressed to infundibuliform; radially appressed-
fibrillose at all times; typically with a metallic luster when fresh; at first
greyish brown (6E2, “Saruk”) fading with age to greyish brown to brown
(6E3-4) with the disc area remaining greyish brown; not hygrophanous nor
translucent-striate; margin incurved becoming plane, entire then eroded;
CONTEXT white to off-white, thickness not measured. LAMELLAE <30 mm
long, 7-10 mm broad; white at first, flesh colored with spore maturation;
uncinate with a decurrent tooth or subdecurrent to decurrent; close; edges
smooth then eroded, not denticulate; wavy, concolorous; lamellulae 4-7 mm
high, 4-15 mm long. STIPE <60 x <14 mm, equal; white becoming at the base
faintly brownish with handling and age; longitudinally appressed fibrillose;
BASAL TOMENTUM absent. Opor strong, ammonia-like when crushed. TASTE
not taken.

BASIDIOSPORES 5 to (rarely) 6 distinct angles in profile and side views,
very rarely 4-angled in polar view; in profile view isodiametric to nearly
heterodiametric, on average subisodiametric; apex acute, obtuse, or flat;
6.9-8.8 x 5.6-7.6 um (x= 8.0 + 0.34 x 6.8 + 0.4 um; Q =1.04-1.4;Q. = 12
+ 0.1; x/y = 64/2). Basrp1a clavate, tapered to a narrow or moderately broad
base, granules abundant but obscure, 26.6-39.9 x 9.9-12.0 um (x = 33. + 3.4 x
11.0 + 0.65 um; Q =2.6-3.6;Q. = 2.6 + 0.3; x/y = 22/2); 4-sterigmate, sterigma
1.2-5.8 um long. LAMELLAR EDGE partially sterile. CHEILOCYSsTIDIA abundant,
but often not on every lamella; appear as terminal cells of tramal hyphae,
versiform in shape (clavate, broadly cylindro-clavate, cylindro-clavate, rarely
vesiculose, sometimes fusiform), short to long, at times at the end of a long
stalk; colorless; 27.6-104.7 x 6.7-28.6 um (x= 53.8 + 16.2 x 13.7 + 4.0 um;

54 ... Largent & Cribari

Q =1.8-9.4;Q. = 4.2 +.1.7; x/y = 61/2). PLEUROCySTIDIA absent. LAMELLAR
TRAMA when sectioned distinct and 20-30 um deep, with the mediostratum
composed of subparallel, longitudinally entangled, dark colored hyphae
bordered by two subhymenia of entangled, colorless, narrow hyphae with
non-gelatinized walls; in squash mounts hyphae narrow to very broad and
with both ends rounded, 59.8-451.0 x 2.3-23.2 um (x = 202 + 110.6 x 10.3
+ 5.1 um; Q =6.1-54.2; Q. = 22.4 + 12.4; x/y = 26/2). PILEIPELLIS ~80 um
deep, a tightly entangled layer of narrow hyphae with distal 1-4 cells erect to
suberect; PILEOCYSTIDIA narrowly clavate to cylindric, 23.4-79.0 x 3.0-12.8
um (x = 44.6 + 15.7 x 7.6 + 2.3 um; Q =2.0-10.6;Q. = 6.3 + 2.4; x/y = 27/2).
PILEAL TRAMAL HYPHAE in squash sections with distal cells with narrow to
broadly acuminate ends, 67.8-261.0 x 8.8-30.6 wm (x = 173.8 + 67.4 x 16.3
+ 7.2 um; Q. = 12.0 + 5.2; x/y = 11/2). STIPITIPELLIs at the apex 33.0-62.3
um deep, an entangled layer of hyphae with erect, suberect, or prostrate distal
cells; CAULOCYSTIDIA clavate to cylindric, 20.5-91.4 x 4.1-10.8 um (x = 49.4
+ 18.2 x 6.7 + 1.7 um; Q =4.4-12.7; Q =7.7 + 3.0; x/y = 23/2). STIPE TRAMAL
HYPHAE in longitudinal sections subparallel and entangled, 103-405 x
6.9-36.8 um (x = 245.2 + 91.0 x 18.6 + 8.2 um; Q = 6.9-25.9;Q. = 14.4 + 5.2;
x/y = 20/2). OLEIFEROUS HYPHAE absent in all tramal tissues. L1IpID GLOBULES
absent. PIGMENTATION slightly to definitely encrusting on lamellar and pileus
tramal hyphae; uniform, cytoplasmic, and light brownish in pileocystidia.
CLAMP CONNECTIONS absent in the pileipellis, typically absent at base of
basidia and cheilocystidia, and absent on the hyphae of the pileipellis.
ECOLOGY & DISTRIBUTION-Gregarious, broadcast over a 10 x 4 m area in
a lawn. Spring; early to late September. Known only from type locality.
ADDITIONAL SPECIMEN EXAMINED—-AUSTRALIA, QUEENSLAND, Cook Region,
Smithfield, 10 Marcia Close, 16.8261°S 145.6857°E, gregarious in lawn, 9 September
2010, Topotype Peter Newling
DISTINCTIVE CHARACTERS-Clitocyboid basidiomata with deeply depressed to
infundibuliform radially appressed-fibrillose greyish brown pilei that have a
metallic luster when fresh, subdecurrent to decurrent close lamellae, an equal
stipe that becomes brownish when handled, a strong ammoniac odor, anda lawn
habitat. Microscopically distinguished by subisodiametric distinctly angular
basidiospores (x = 9 x 7 um), versiform often long-stalked cheilocystidia, a
pileipellis ~80 um deep with a tightly entangled layer of narrow hyphae with the
distal 1-4 cells erect to suberect, cytoplasmic pigmentation in the pileocystidia
and encrusting pigment on the walls of the pileal and lamellar tramal hyphae
and the presence of caulocystidia.

Clitopiloides & Trichopilus spp. nov. (Australia) ... 55

Fig. 1. Clitopiloides prati (holotype HSC A1429; except B): A. basidioma with deeply depressed
to nearly infundibuliform pileus; B. (Topotype HSC A1430) overly mature basidiomata with
infundibuliform pileus; C. 5-angled basidiospores; D. clavate sterigmate basidium tapered to
narrow base; E. cluster of broadly clavate to cylindro-clavate cheilocystidia; F. tightly entangled
stipitipellis ~2.4 cm down stipe apex, with semi-erect to prostrate caulocystidia; G. pileipellis
from disc with loosely entangled hyphal layer with semi-erect cylindro-clavate pileocystidia.
Scale bars: A, B = 14mm; C = 8 um; D= 33 um; E = 15 um; F = 50 um; G = 50 um.

56 ... Largent & Cribari

CoMMENTS~Clitopiloides prati morphologically resembles C. cyathus
(Romagn. & Gilles) Largent from Gabon and the Ivory Coast in its brownish
colored basidiomata, deeply depressed to infundibuliform pileus, decurrent
lamellae, presence of cheilocystidia, and 5-6-angled basidiospores measuring
7-9 x 5.7-7.6 um. However, C. cyathus is distinguished by its yellowish brown
(“bistre”) glabrous, translucent-striate, hygrophanous pileus, denticulated
lamellae, clavate stipe, lack of odor or an odor of bitter almonds, cheilocystidia
measuring 25-45 x (8.5-)12-16(-24) um, vacuolar pigmentation, and
rainforest habitat (Romagn. & Gilles, 1979). Entoloma cuboidosporum (Beeli)
E. Horak from Malaysia, Singapore, Zaire, Madagascar, Gabon, and the Congo
shares the brownish, deeply depressed to infundibuliform pileus and decurrent
lamellae found in C. prati but differs in its glabrous pileus, farinaceous odor,
and cuboid basidiospores (Horak, 1980).

GenBank’s Blastn tool shows the ITS sequence from C. prati as 88.63%
similar to Entoloma subclitocyboides W.M. Zhang (= E. subinfundibuliforme
T.H. Li & Chuan H. Li) from China. Entoloma subclitocyboides differs from
C. prati in its broader (7.0-8.5 um) isodiametric basidiospores, dirty yellowish
to pale yellow brown pileus, adnexed subventricose lamellae, lack of odor, lack
of encrusting pigments in the lamellar trama and pileipellis, presence of clamp
connections, and habit on soil in mixed forest (He & al. 2014).

Trichopilus lecythiformis Largent, sp. nov. Fig, 2
IF 558178

Differs from Trichopilus tibiiformis by its yellowish brown, translucent striate pileus,
a stipe that bruises yellow brown, larger basidiospores, and abundant lecythiform
cheilocystidia and pleurocystidia.

Type: Australia, New South Wales, North Coast, Myall Lakes National Park, Mungo
Brush campground 32.5457°S 152.3094°E, 30 April 2012, DL Largent 10482 (Holotype,
DAR 81840).

EryMoLoGy—lecythiformis (Latin) referring to the lecythiform cheilocystidia.

BASIDIOMATA omphalinoid. Prteus 13-14 mm broad x 1.0-1.5 mm tall,
convex to plano-convex, shallowly depressed, tomentulose on the disc,
glabrous elsewhere; dark yellowish brown (5F4 “sepia brown”) in center at all
times, elsewhere light yellowish brown (5E4 “hair brown”) becoming darker
with age and drying (5F7 “coffee”), margin decurved then plane, very slightly
crenulate, translucent striate to the disc, moist and wet, not hygrophanous.
LAMELLAE 6 mm long x 2-2.5 mm high, adnate to short decurrent at first, then
long decurrent, at first off-white to orange white (5A4), becoming pale orange
(5B3) with sporulation, narrow then distant, edge smooth and concolorous;

Clitopiloides & Trichopilus spp. nov. (Australia) ... 57

in profile view; C. clavate sterigmate basidia tapered to narrow bases; D. tibiiform and lecythiform
cheilocystidia; E. clavate pileocystidia with plasmatic brown pigments; F. disc pileipellis showing
loosely entangled palisadoderm. Scale bars: A = 14 mm; B = 8 um; C = 12 um; D = 14 um; E = 13
um; G = 55 um.

58 ... Largent & Cribari

lamellae 2-3 between lamellae, in 2 series (one short, one medium long). STIPE
22-28 x 1.0-1.5 mm, straight, equal, faintly pruinose at the apex, elsewhere
glabrous, yellowish white (4A2) to light yellowish grey (5B2 “putty”), bruising
yellowish brown (~5E5 “bronze” = “bronze brown’); BASAL TOMENTUM absent.
Opor and Taste mild, not distinctive.

BASIDIOSPORES 5-6-angled, angles distinct, hilar appendage large and
distinct, apex rarely acute, often obtuse, nearly all spores with single globule, on
average heterodiametric, 9.8-14.4 x 6.8-9.9 wm (x = 11.3 + 1.0 x 8.3 + 0.67 wm;
Q = 1.19-1.55;Q. = 1.35 £ 0.10; x/y = 53/1). Basip1a clavate, narrowly tapered
at the very base (7/8 inflated), with granules that are not brilliant; 23.0-36.1
x 11.2-13.8 um (x = 29.3 + 2.9 x 12.4 + 0.85 um; Q =1.80-2.76; Q., = 2.38
+ 0.26; x/y = 16/1)); 4-sterigmate, sterigma 2.52-5.79 um. CHEILOCYSTIDIA
abundant, lamellar edge mostly sterile, tibiiform and lecythiform, colorless,
29.7-57.0 x 10.2-17.4 um (x = 46.7 + 7.8 x 14.2 + 1.5 pm; Q =2.5-4.0;
Q_=3.29+0.45;x/y=19/1);head4.0—-9.0 um wide (x/y21/1);neck2.4—6.2 um wide
(x/y = 21/1). PLEUROCysTIDIA rare at the lamellar edge, similar in shape and
size to cheilocystidia. LAMELLAR TRAMAL HYPHAE subparallel and in squash
mounts rounded at both ends, 55.3-100.9 x 12.2-20.6 wm (x = 78.8 + 13.9 x 15.1
+ 3.1 um; Q =3.50-6.75; Q. = 5.35 + 1.13; x/y = 7/1). PILEIPELLIs on the disc a
distinct palisadoderm with erect hyphal elements attached laterally, distal 4-5
cells inflated, prostrate from the pileus middle to margin; pileocystidia clavate
to cylindro-clavate, 27.1-109.3 x 8.25-18.8 um (x = 56.7 + 20.4 x 13.9 + 2.7 um;
Q = 2.62-8.11; Q. = 4.44 + 1.44; x/y = 38/1). PILEAL TRAMAL HYPHAE
subparallel and entangled, 30.8-56.0 x 5.7-9.5 um (x = 47.0 + 8.9 X 8.6 + 1.5 um;
Q =3.41-8.80; Q =5.66 + 1.78; X/Y = 6/1). STIPITIPELLIS a cutis; CAULOCYSTIDIA
absent. STIPE TRAMAL HYPHAE in longitudinal section, subparallel and
entangled; 55.6-273.3 x 7.4-28.5 um (x = 155.4 + 59.3 x 15.2 + 5.0 um;
Q = 4.83-19.40; Q. = 10.74 + 4.35; x/y = 15/1), OLEIFEROUS HYPHAE absent
in all tramal tissues. Lip1D GLOBULES absent. PIGMENTATION cytoplasmic,
uniform, brown in apical cells of the pileipellis, encrusted in the basal cells;
basidia containing non-brilliant granules. CLAMP CONNECTIONS absent.

ECOLOGY & DISTRIBUTION- Scattered in grass along the beach track,
Myall Lakes National Park, coastal New South Wales, Australia, in late April
(autumn). Known only from the type locality.

ComMEnts-Trichopilus lecythiformis is known from a single collection of a
single basidiome from sandy soil in Myall Lakes National Park in late April.
However, this species is distinct from all other Australian entolomatoid
fungi due to its combination of small delicate basidiomata, pileus with a

Clitopiloides & Trichopilus spp. nov. (Australia) ... 59

yellowish brown disc, long clavate to cylindro-clavate pileocystidia, 5-6-sided
basidiospores measuring ~11.3 x 8.3 um, tibiiform to lecythiform colorless
cheilocystidia and pleurocystidia, palisadoderm pileipellis, and the absence of
caulocystidia and clamp connections. As far as I know, this the first report of a
Trichopilus species in Australia with a small delicate basidiome.

Seven other species might be confused with T! lecythiformis due to their
small delicate basidiomata and tibiiform/lecythiform cheilocystidia. They
can be distinguished from T: lecythiformis as follows: T: tibiiformis Largent &
Aime from Guyana has a dark violet brown shaggy, opaque pileus and smaller
(7.6-9.8 x 4.5-6.7 um) 6-7-angled basidiospores (Aime & Largent 2010).
Entoloma mariae G. Stev. from New Zealand has smaller (9-11 x 7-8.5 um)
basidiospores, larger (35-100 x 10-22 um) cheilocystidia, a pruinose stipe,
and clamp connections; E. perplexum E. Horak, also from New Zealand, has
a brownish grey small (5-10 mm diam) pileus, cinnamon-colored young
lamellae, a pileipellis composed of vesiculose or clavate capitate pileocystidia,
a weak farinaceous taste and odor, and 5-6-angled basidiospores measuring
10-13 x 7-9 um (Horak 2008). Entoloma festivum Noordel. & al. from the
Netherlands has a warm reddish brown hygrophanous pileus with a black
disc, smaller (8.5-10.5 x 5.5-7.5 um) 5-8-angled basidiospores, cheilocystidia
with a vacuolar pigment, and clamp connections (Noordel. & al. 2010).
Rhodophyllus capitatus Romagn. & Gilles has a pinkish white, scaly, translucent
striate pileus, pale brownish lamellae, smaller (7-8.5 x 5.7-6.7-4m) 4-5-angled
prismatic basidiospores; R. applanatus Romagn. & Gilles has a fairly dark
pinkish brown, translucent-striate scaly pileus, smaller (7-8.5 x 5.7-6.7 um)
5-6-angled basidiospores; and R. lepiotoides Romagn. & Gilles has a yellow
brown opaque pileus with a brown disc and slight umbo and smaller (6.5-7.5
x 5.7-6.5) 4-5-angled basidiospores (Romagn. & Gilles 1979). In the literature
cited above, there has only been one collection studied for five of these species
and two collections for R. lepiotoides, suggesting that these species are either
rare or the small basidiomata overlooked in the field.

Unfortunately, because all attempts at obtaining DNA sequences from DLL
10482 (the holotype of T! lecythiformis) were unsuccessful, this collection is
proposed as a Trichopilus species based on morphology alone. However,
phylogenetic analyses using four genes demonstrated that Trichopilus is a
statistically supported genus with bootstrap values of 100%/1 representing
a monophyletic group nested deep within the Inocephalus—Cyanula clade.
Sequences for all four genes (mtSSU, nLSU, rrpb2, and tefl) were obtained
from T. tibiiformis with delicate basidiomata in this study (Karstedt & al. 2019).

60 ... Largent & Cribari

We are confident that if sequences are obtained from future collections of
T. lecythiformis, the species will cluster in this well-supported group.

Acknowledgments

Materials required to complete this manuscript were provided by the Largent
family trust. The Australian Tropical Herbarium and the School of Marine and
Tropical Biology, James Cook University, provided fieldwork and logistical support.
The DNA sequences generated in this study were completed and financed by Dr. Sarah
Bergemann. Comments by the two expert reviewers, Dr. Sarah Bergemann (Middle
Tennessee State University, Murfreesboro, TN, USA) and Dr. Timothy J. Baroni (New
York State University at Cortland, NY, USA), and by the Nomenclature Editor, Dr.
Shaun Pennycook, were also helpful. We wish to thank Peter Newling for his assistance
in collecting in northeastern Queensland and collecting PN40, the holotype collection
for Clitopiloides prati. We also wish to thank Pam O'Sullivan and Skye Moore for
their assistance in collecting in New South Wales. Finally, David and Pamela Largent
especially wish to thank Dr. Sandra Abell for being an extraordinary research advisor
and contact at James Cook University during the five years of research in northeastern
Queensland.

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species from New South Wales and Queensland, Australia. Mycotaxon 125: 11-35.
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Largent DL, Kluting KL, Anderson NM, Bergemann SE. 2016. New leptonioid species from
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Noordeloos ME, Rommelaars LCAF, Gelderblom JNJ. 2010. Entoloma festivum, a new
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MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 63-71
https://doi.org/10.5248/137.63

Neoacrodictys elegans gen. & sp. nov.
from Hainan Province, China

JI-WEN X14, TAI-CHANG Mu, ZHAO-XUE ZHANG,
ZHUANG Li, XIU-GUO ZHANG*

Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests,
College of Plant Protection, Shandong Agricultural University,
Taian, Shandong 271018, China

“CORRESPONDENCE TO: sdau613@163.com

ABSTRACT—A new anamorphic genus and species, Neoacrodictys elegans, is illustrated and
described from dead branches of an unidentified plant in Hainan Province, China. The genus
is characterized by darkly pigmented turbinate to obpyriform muriform conidia produced
from monoblastic integrated terminal conidiogenous cells on macronematous unbranched
conidiophores.

KEY worDS—asexual Ascomycota, hyphomycetes, saprobes, taxonomy

Introduction

During our continuing surveys of dematiaceous hyphomycetes colonizing
diverse plant habitats from the forests of Hainan, China, a fungus that
does not match any existing genera was collected growing on unidentified
dead twigs. Morphological studies and a literature review (Ellis 1971,
1976; Subramanian 1971; Matsushima 1975, 1983, 1985, 1989, 1993, 1995;
Castafteda-Ruiz 1986; Castafeda-Ruiz & Kendrick 1990a,b, 1991; Wu &
Zhuang 2005; Seifert & al. 2011; Ma & al. 2016, 2021; Xia & al. 2016, 2017;
Xu & al. 2020a,b, 2021; Zhang & al. 2020; Niu & al. 2021) indicated that the
fungus represents an undescribed genus. Molecular phylogenetic analysis
confirmed its morphological identity, and the fungus is described here as a
new genus and species.

64 ... Xia & al.
Materials & methods

Isolates & morphological analysis

Samples of dead branches collected were placed in separate zip-lock plastic bags,
taken to the laboratory, and then incubated at 27 °C for more than 2 weeks in an
artificial climate box in 9 cm diameter plastic Petri dishes with moistened filter paper.
Single-spore cultures of hyphomycetes that could not be identified on natural substrate
were isolated from dead branches and incubated on potato-dextrose agar (PDA: 200
g boiled and filtered white potatoes, 20 g dextrose, 15 g agar, 1 L distilled water). All
PDA plates were incubated at 25 °C for 1-2 months. The colonies were photographed
using a Powershot G7X mark II digital camera. Micromorphological characters were
observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope,
both fitted with Olympus DP80 high definition colour digital cameras. All fungal
strains were stored in 10% sterilized glycerin at 4 °C for further studies. The specimens
are deposited in the Herbarium of Plant Pathology, Shandong Agricultural University,
Taian, Shandong, China (HSAUP). Ex-type cultures are deposited in the Shandong
Agricultural University Culture Collection, Taian, Shandong, China (SAUCC).

DNA extraction, PCR amplification, sequencing

Genomic DNA was extracted from colonies grown on PDA, using the CTAB
method (Doyle & Doyle 1990). The large subunit ribosomal RNA gene (LSU) was
amplified and sequenced by using primers pairs LROR/LRS (Vilgalys & Hester 1990,
Glass & Donaldson 1995).

PCR was conducted using an Eppendorf Master Thermocycler. DNA was amplified
in 25-uL volumes containing 12.5 uL Vazyme Green Taq Mix, 1 uL of each forward
and reverse primer (10 uM) (Biosune), and 1 uL template genomic DNA adjusted to
a total volume of 25 uL with distilled deionized water. PCR parameters were 95 °C
for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C
for 30 s, and extension at 72 °C for 1 min, ending with a final elongation step at 72 °C
for 10 min. PCR products were estimated visually by staining with GelRed after 1%
agarose gel electrophoresis. Sequencing was done bi-directionally by Biosune Co. Ltd.
(Shanghai, China). Consensus sequences were obtained using MEGA 7 (Kumar & al.
2016). All sequences generated in this study were deposited in GenBank (TaBLE 1).

Sequence alignment and phylogenetic analysis

The quality of our amplified nucleotide sequences was checked and combined
by MEGA 7 (Kumar & al. 2016), and reference sequences were retrieved from the
National Center for Biotechnology Information (NCBI). Sequences were aligned
using MAFFT 7.310 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh & al.
2019), and manually corrected using MEGA 7.

The LSU sequences were analyzed phylogenetically using Maximum-Likelihood
(ML) and Bayesian Inference (BI) methods. RaxML and Bayesian analyses were run
on the CIPRES Science Gateway portal (Miller & al. 2012) using RaxML 8.2.9 and
MrBayes 3.2.6. Evolutionary models were calculated using MrModelTest 2.3 (Nylander

TABLE 1. Strains and sequences included in the phylogenetic analyses.

Neoacrodictys elegans gen. & sp. nov. (China) ... 65

The new sequence is set in bold.

TAXON

Acrodictys bambusicola
Acrodictys elaeidicola
Asterina sp.
Botryotinia fuckeliana
Buelliella physciicola
Buelliella poetschii
Junewangia lamma
Junewangia sphaerospora
Karschia cezannei
Karschia talcophila

Labrocarpon canariense

‘Melaspilea lekae

Mycosphaerella pneumatophorae

Mycosphaerellaceae sp.

Melaspileopsis cf. diplasiospora

Melaspileopsis sp.

Neoacrodictys elegans

Rhexoacrodictys erecta

Rhexoacrodictys fimicola

Stictographa lentiginosa

VOUCHER

CGMCC 3.18641

CGMCC 3.18642

CGMCC 3.18643

SH-2014

AFTOL-ID 59

Ertz 18113 (BR)

Ertz 19173 (BR)

Ertz 18115 (BR)

Ertz 18116 (BR)

CGMCC 3.18652

CGMCC 3.18653

CGMCC 3.18655
Cezanne-Eichler B26 (hb. Diederich)
Cezanne-Eichler 7453 (hb. Diederich)
Ertz 19186 (BR)

Diederich 16749 (hb. Diederich)
Ertz 16308 (BR)

Ertz 16907 (BR)

Ertz 17325 (BR)
JK5253B=AFTOL-ID 762
SD-01

KH00300

Ertz 16247 (BR)

Ertz 16624 (BR)

Ertz 16625 (BR)

Ertz 17904 (BR)

Ertz 17913 (BR)

SAUCC H4600

CGMCC 3.18656

CGMCC 3.18657

CGMCC 3.18658

CGMCC 3.18660

Ertz 17447 (BR)

Ertz 17570 (BR)

van den Boom 47621 (hb. v.d. Boom)

GENBANK LSU
KX033564
KX033568
KX033569
KM386978
AY544651
KP456147
KP456148
KP456149
KP456150
KU751882
KU751883
KX033572
KP456152
KP456153
KP456154
KP456155
KP456157
KP456158
KP456162
FJ176856
JN872645
GU017553
KP456164
KP456165
KP456166
KP456167
KP456168
MW907608
KX033555
KX033556
KX033553
KX033554
KP456169
KP456170
KP456171

66 ... Xia & al.

2004) to select the best-fit model for each data partition according to the Akaike
criterion. For ML analyses the default parameters were used and bootstrap support
(BS) was calculated using the rapid bootstrapping algorithm with the automatic halt
option. Bayesian analyses included two parallel runs of 5,000,000 generations, with
the stop rule option and a sampling frequency set to each 1000 generations. The 50%
majority rule consensus trees and posterior probability (PP) values were calculated
after discarding the first 25% of the samples as burn-in. Trees were plotted in FigTree
1.4.2 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS 5.

Phylogenetic results

The dataset comprised 35 taxa representing 17 named species and four
undetermined species including Botryotinia fuckeliana (AFTOL-ID 59) as
outgroup. The final LSU alignment totaled 1178 characters including gaps.
Of these characters, 825 were constant, 89 parsimony-uninformative, and
264 parsimony-informative. For the BI and ML analyses, the substitution
model GTR+I+G for LSU was selected and incorporated into the analyses.
The topology of the ML tree confirmed the tree topologies obtained from
BI analyses, so only the ML tree is presented (Fic. 1). In this tree, our strain
formed an independent clade.

Taxonomy

Neoacrodictys J.W. Xia & X.G. Zhang, gen. nov.
MB 816515

Differs from Acrodictys by its turbinate conidia and oblique septa that are often marked
by dark bands.

TYPE SPECIES: Neoacrodictys elegans J.W. Xia & X.G. Zhang

ErymMo.Loecy: Neoacrodictys = “neo-” + “acrodictys” (Lat.), referring to its similarity to

the genus Acrodictys.
CONIDIOPHORES macronematous, mononematous, unbranched, septate,
brown to dark brown, paler towards the apex, indeterminate with flask-shaped
percurrent extensions. CONIDIOGENOUS CELLS monoblastic, integrated,
terminal, cylindrical, pale brown to brown, smooth. Conidial secession
schizolytic. Conrpia solitary, acrogenous, muriform, turbinate to obpyriform,
euseptate, with one longitudinal and a few transverse oblique septa usually
obscured by dark bands.

Neoacrodictys elegans J.W. Xia & X.G. Zhang, sp. nov. FIG. 2
MB 816516

Differs from Acrodictys spp. by its turbinate conidia and oblique septa usually obscured
by dark bands.

Neoacrodictys elegans gen. & sp. nov. (China) ... 67

Botryotinia fuckeliana

0.07

Fic. 1. Phylogram generated from RaxML analysis based on the LSU gene. Bootstrap support
values are shown for ML >70% and BI >0.85. Branches shortened to fit the page are indicated by
two diagonal lines accompanied by the length reduction factor. The new sequence is indicated
in bold.

Type: China, Hainan Province: Ledong, Jianfengling National Forest Park, 18.70°N
108.87°E, on dead branches of an unidentified broadleaf tree, 22 April 2014, Y.R. Ma
(Holotype, HSAUP H4600; ex-type culture, SAUCC H4600; GenBank MW907608).

EryMo_oey: refers to its conidia that have an elegant appearance.

COLONIES effuse, brown, hairy. MycEe.ium partly superficial, partly immersed
in the substrate. CONIDIOPHORES macronematous, mononematous, erect,
unbranched, straight or flexuous, thick-walled, smooth, dark brown; swollen
at the base, narrower and paler toward the apex, 3-7-septate, 54-105 x
4.5-7.5 um at the broadest part, indeterminate with 1-2 flask-shaped percurrent

68 ... Xia & al.

extensions. CONIDIOGENOUS CELLS integrated, terminal, cylindrical to
doliiform, subhyaline to pale brown, smooth, monoblastic; extending through
the scar of the last conidium on the conidiophores. Conip1a solitary, muriform,
turbinate to obpyriform, 15.5-22.5 x 12-18 um, with 1 or 2 transverse septa
and 1 or 2 longitudinal or oblique septa; septa typically crossing at right angles
and usually obscured by a black band. Basal cell funnel-shaped, delimited by a
transverse septum, pale brown. Conidial secession schizolytic.

Discussion

Neoacrodictys demonstrates unique morphological and ontogenetic
features. It is distinguished by macronematous, mononematous, cylindrical
and unbranched conidiophores with holoblastic conidiogenous cells that
produce darkly pigmented, muriform, turbinate to obpyriform conidia.
A key to Neoacrodictys and morphologically similar genera is provided.

Neoacrodictys elegans resembles Acrodictys bambusicola M.B. Ellis,
A. elaeidicola M.B. Ellis, and A. malabarica Subram. & Bhat, which differ by
conidial shape (Ellis 1961, Subramanian & Bhat 1989, Xia & al. 2017). Also,
the genera Acrodictys and Neoacrodictys belong in different phylogenetic
clades (Fic. 1).

Key to Neoacrodictys and morphologically similar genera

LEG onidtadbicellwlar as Ait.28 Bit.0e Pee BEN WEE LE Wet ee Ityorhoptrum
Ie@Conidia MUPTOCM: 5... gdb adis gdh edd needs neegd badd oeaw bow dibeeie 4d heres Z
2} CONIA WIEN APPCAGASES ssliez sh ires-z chipsen-agub shea gih sete gr eemengh idea gst Pseudoacrodictys
2 AOhidia Without ap PelidaGes wr. c.s sso. rates sta ssi stadt ora cet aed aed Bas 3
3-Conidial secession thexOlytie wh cede eontegt 8 mcwitege B noeceye B heenegd # nutes Rhexoacrodictys
SECOnidial secessign scMiZ@lylie WF 1089 Feats F188 UBB ok WiaBDeh aA COL Pagel SS 4
4, Genidia Inaturing ahter SCCESSION f.i.5.. Wheaton ea iewilerd ste are edd areas Acrodictyella
4, Conidia becoming pigmented and septate prior to secession ................44. 5
5. Conidial midpoint attached to the conidiogenous cell ........... Coleodictyospora
5. Conidial base attached to the conidiogenous cell ............ 0... cee eee eee eee 6
6. Conidiophore aseptate, comprising a single conidiogenous cell

that extends percurrently after each conidial dehiscence .......... Junewangia
6. Conidiophores conspicuously septate, a new conidiogenous cell

forming: atter each conidial dehiscence 2. fh. wiucbes wboade x wautbes aude ante lt 7
7. Conidial basal cell inconspicuous,

reduced to-a:short;-cylindricalsdisk § sav. qay uw See ewe u eee ve Acrodictys
7. Conidial basal cell conspicuous,

distinctly funnel-shaped and flat at the base.................... Neoacrodictys

Neoacrodictys elegans gen. & sp. nov. (China) ... 69

Fic. 2. Neoacrodictys elegans (holotype, HSAUP H4600): a. Colony on PDA (surface and reverse);
b. Colony on MEA (surface and reverse); c. Conidia; d—f. Conidiophores, conidiogenous cells,
and conidia. Scale bars: c—f = 20 um.

Acknowledgments

The authors express gratitude to Dr. Jian Ma (College of Agronomy, Jiangxi
Agricultural University, Nanchang, Jiangxi, China) and Dr. Li-Guo Ma (Institute
of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, Shandong,

70 ... Xia & al.

China) for serving as pre-submission reviewers and to Dr. Shaun Pennycook for
nomenclatural review and Dr. Lorelei L. Norvell for editorial review. This work
was jointly supported by the National Natural Science Foundation of China (Nos.
31900014, U2002203, 31750001) and National Science and Technology Fundamental
Resources Investigation Program of China (2019FY 100700).

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MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 73-87
https://doi.org/10.5248/137.73

Neotypification of Claviceps humidiphila
and recognition of C. bavariensis sp. nov.

M1Ao Liv", Eyt TANAKA?,, MIROSLAV KOLARIK?

‘Ottawa Research and Development Centre, Agriculture and Agri-Food Canada,
960 Carling Ave. Ottawa, Ontario K1A0C6 Canada

? Ishikawa Prefectural University,
1-308 Suematsu, Nonoichi, Ishikawa 921-8836 Japan

* Department of Botany Faculty of Science, Charles University,
Benatska 2CZ-128 01, Praha 2 Czech Republic

“CORRESPONDENCE TO: miaomindy.liu@agr.gc.ca; tanakae@ishikawa-pu.ac.jp

ABSTRACT—Claviceps humidiphila |= C. purpurea var. phalaridis] was previously typified with
the holotype collected by Tanda in Japan and an epitype from Bavaria, Germany. Phylogenetic
analyses based on translation elongation factor 1-a (TEF1l-a) and RNA polymerase II second
largest subunit (RPB2) indicated that the previously designated epitype from Germany was
not conspecific with the Japanese species. The German specimen is proposed as a new species,
C. bavariensis, and a specimen collected from the type location (Chiba, Japan) is designated
as a neotype for C. humidiphila, replacing the lost holotype.

Key worps—Ascomycota, Clavicipitaceae, ergot fungi, Hypocreales, taxonomy

Introduction

Claviceps was validly published by Tulasne (1853), as discussed by Donk
(1963). Claviceps purpurea Tul. was selected as lectotype by Clements &
Shear (1931) and accepted by Donk (1963). Tulasne (1853) noted the hosts
of C. purpurea include cereal crops (e.g., Avena, Secale, Triticum) and grasses
(e.g., Alopecurus, Dactylis, Lolium, Poa). Thereafter, the reported host range
increased to more than 400 species in Poaceae. Molecular studies (Pazoutova
& al. 2000, Douhan & al. 2008) revealed cryptic speciation within the pre-
molecular concept of C. purpurea s.lat. Pazoutova & al. (2015) provided

7A ... Liu, Tanaka, Kolatik

formal taxonomic descriptions for four phylogenetic species to replace
C. purpurea s.lat.: C. purpurea s.str., C. arundinis, C. humidiphila, and
C. spartinae.

Claviceps humidiphila was characterized by its frequently larger conidia
than C. purpurea, sclerotia capable of floating on water, and typical habit on
Calamagrostis, Deschampsia caespitosa, and Phalaris arundinacea. Previously,
Tanda recognized three varieties: C. purpurea var. alopecuri, C. purpurea
var. dactylidis, and C. purpurea var. phalaridis, producing conspicuously
larger conidia and on specific host plants. Claviceps purpurea var. alopecuri
was found on Alopecurus, but also on Agrostis clavata, A. palustris,
Calamagrostis epigejos, C. hakonensis, Holcus lanatus, Phleum pratense, Poa
spp., Polypogon fugax, and Trisetum bifidum (Tanda 1978a,b,c, 1979a,c,
1981; Tanda & Kawatani 1980). Claviceps purpurea var. dactylidis was found
on Dactylis glomerata and Calamagrostis epigejos (Tanda 1980). Claviceps
purpurea var. phalaridis occurred on Phalaris, Calamagrostis epigejos, and
C. pseudophragmites (Tanda 1979a).

Pazoutova & al. (2015), who noted that the conidial dimension and host
range of C. purpurea var. dactylidis and C. purpurea var. alopecuri overlapped
with both C. purpurea s.str. and C. humidiphila, treated the varieties as taxa
of uncertain identities. However, C. purpurea var. phalaridis was considered
as representing the same taxon as the species later named C. humidiphila.
The name C. purpurea var. phalaridis could not be elevated to species rank
as a stat. nov. because this would create an illegitimate later homonym of
C. phalaridis J. Walker [= Aciculosporium phalaridis (J. Walker) M. Kolarik &
Pichova]. Instead, Claviceps humidiphila was proposed as a nom. nov. based
on the replaced synonym Claviceps purpurea var. phalaridis. Tanda (1979b)
designated a holotype from Chiba, Japan, now believed to have been lost
(see TaxONOMY: COMMENTS below, p. 78). Pazoutova & al. (2015) designated
an epitype from Bavaria, Germany, but phylogenetic analysis indicates that
the epitytpe is not conspecific with Japanese collections. Here, we propose
Claviceps bavariensis as a new species holotypified by the Bavarian collection
and designate a recent specimen from Chiba, Japan, as the neotype of
C. purpurea var. phalaridis [= C. humidiphila].

Material & methods

DNA extraction, PCR, sequencing, analyses
Axenic cultures were developed for two samples collected from Canada (Alberta)
and gDNA extracted following Shoukouhi & al. (2019). For 29 samples from Japan

Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 75

(Chiba, Gifu, Ishikawa, Kanagawa, Miyagi, Nagano, Niigata, Tochigi, Toyama), axenic
cultures were developed from peeled ergots that were surface-disinfected in 2%
sodium hypochlorite solution for 1 min; gDNA was extracted according to Izumitsu
& al. (2012). Two genomic regions, RPB2 (RNA polymerase II second largest subunit)
and TEF1-a (translation elongation factor 1-a), were amplified and sequenced using
modified fRPB2-5F (5’-rTTCGTGGTATTGTTCGCAGA-3’) specific for ergot fungi
(Pazoutova & al. 2015) and fRPB2-7cR (Liu & al. 1999), and the primer pair EF1-983F
and EF1-2218R (Rehner & Buckley 2005). For 29 samples from Japan, the RPB2 regions
were amplified and sequenced using Cla-RPB2 (5’-CAGTGAAACCAGAAAGGCCTTC-3’)
and modified fRPB2-7cR (5’-CCCATGGCCTGCTTACCCAT-3’). Polymerase chain
reactions (PCRs) were performed in 10 uL volumes containing final concentrations
of 10x Titanium Taq buffer (with 3.5 mM MgCl), 0.1 mM dNTPs, 0.08 uM each of
forward and reverse primer, 50x Titanium Taq polymerase (Takara Bio, California),
0.01 mg BSA, and 1 uwL of DNA template. A touchdown protocol was applied with
an initial denaturation at 95 °C for 3 min, followed by 5 cycles of 95 °C for 1 min,
annealing at 63 °C (decrease 1 °C per cycle) for 45 s, and extension at 72 °C for 1 min
30 s followed by 30 cycles of 95 °C for 1 min, annealing at 58 °C for 45 s, extension at 72
°C for 1 min 30 s, concluding with a final extension at 72 °C for 8 min. PCR products
were sequenced using a ABI BigDye Terminator 3.1 cycling sequencing kit on Applied
Biosciences Prism 3130xl Genetic Analyzer.

Fifty-nine reference RPB2 and TEFl-a sequences from previous studies
downloaded from GenBank were compiled and aligned with sequences generated
in the present study (31 strains) using MAFFT online version 7 (Katoh & al. 2019,
https://mafft.cbrc.jp/alignment/server/, accessed in Aug 2020). Alignments
of concatenated sequences were generated in Geneious Prime 2020.1.2.
(http://www.geneious.com). Maximum parsimony (MP) analyses were performed
using PAUP* 4.0b10 (Swofford 2002). Heuristic searches with 200 replicates of
random stepwise addition and tree bisection-reconnection branch swapping were
conducted with a limit of 1,000,000 re-arrangements for each replicate. Bootstrapping
analyses used 2000 replicates of a full heuristic search with random stepwise addition
of 20 replicates with limit of 50,000 rearrangements per replicate. Bayesian inference
analyses were conducted using MrBayes 3.2 (Ronquist & al. 2012) with a GTR
model chosen by a previous study (Liu & al. 2021). Each run was set to four chains
of 100,000,000 MCMC generations, sampling frequency was every 2000 generations,
terminated when the standard deviation of the average split frequency was lower than
0.01. The BI consensus tree was directly generated after 25% burn-in.

Morphological examination

Specimens were conserved in the Herbarium, Department of Botany, National
Museum of Nature and Science, Tsukuba, Japan (TNS) and the Canadian National
Mycological Herbarium (DAOM); cultures were conserved in NARO Genebank,

76 ... Liu, Tanaka, Kolatik

Microorganism Section, Genetic Resources Center, National Agriculture and Food
Research Organization, Tsukuba, Japan (MAFF).

Sclerotia were tested for floating ability using the protocol developed by Pazoutova
& al. (2000). The shape, size, color, and surface of sclerotia were recorded from
observations of available sclerotia. Specimens were photographed using a DFC425
camera and Leica Application Suite 4.12.0 software attached to Leica M165C or Motic
SMZ-168-BL stereo microscopes. Colors were characterized using html color HEX
codes at the maximum approximation (https://htmlcolorcodes.com/color-names/).
Sclerotial tissues were sectioned by hand for microscopical examination. Conidia
were washed off from sclerotial surfaces and mounted in water or lactic acid for
examination using a Zeiss Axio Scope.Al microscope with differential interference
contrast (DIC) illumination, or Zeiss Imager M2. Microphotographs were taken
with either a Jenoptik ProgRes SpeedXTcore 5 digital camera using ProgRes image
processing software for CCC samples or with (alternatively) an Axiocam 503 color
camera using a Zeiss ZEN (blue edition) 2.6 pro imaging processing for Canadian
samples, and for Japanese samples a MicroPublisher 5.0 or 3.3 RTV using QImaging
QCapture Pro software or a WRAYCAM-NOA2000 using the Wraymer MicroStudio
processing package. Conidiogenesis in sclerotia was observed under a Zeiss Imager
M2 by mounting small fragments of internal sclerotial tissue obtained by slicing or
picking.

Germinated sclerotia were obtained by chill-treating sclerotia on moistened quartz
sand in sealed cups at 4°C about 3 months followed by incubation under 12 h light/12 h
dark condition at 20 °C for a few weeks. Ascostromal sections were prepared as
described by Tanaka & al. (2020). Examination and microphotography of asci and
ascospores followed the same procedures as used for conidia.

Phylogenetic results

DNA sequences generated in this study were submitted to GenBank.
Including 59 reference sequences, the TEFl-a and RPB2 matrices
comprised 90 taxa and 918 and 1027 characters. The phylogenetic tree
based on the concatenated alignment of TEFl-a and RPB2 placed the
specimen collected from the type location of C. purpurea var. phalaridis
(TNS-F-60506 on Phalaris arundinacea Chiba) in a different clade,
sister to CCC 434 (= DAOMC 251717), the epitype from Germany.
Here, we retain the name C. humidiphila for the Japanese clade and
propose C. bavariensis for the German clade. The two are sister
species and closely related to C. arundinis and C. perihumidiphila
(Fic. 1). Phylogenetic trees based on the individual genes showed localized
low resolution as discussed by Shoukouhi & al. (2019 and Liu & al. (2020).

C. citrina CCC 265
100/

100/1

C. sect. Claviceps

Epichloe bromicola AL0434
Epichloe glyceriae E277

Claviceps bavariensis sp. nov. & C. humidiphila neotypified ...

C. sect. Pusillae

C. paspali CCC 130

99/1 C. occidentalis DAOMC 250578
C. quebecensis DAOMC 2518985"
7510.98— ¢. cyperi CCC 1219
roel C. fimbristyliais CCC 14726"
‘ C. nigricans CCC 802
C. grohii CBS124x47
7110.95;— ©. capensis CCC 1504
oe C. macroura CCC 1482&T
C. pazoutovae CCC 1485"
p8/1 C. monticola CCC 1483
oot C. purpurea DAOMC 250822
C. purpurea DAOMC 251723°T
C. purpurea DAOMC 250877
[__ C. zizaniae DAOMC 252145
93/1 C. ripicola DAOMC 251844€T
C. ripicola DAOMC 252149
C. ripicola DAOMC 251923
C. spartinae DAOMC 251720
400/1 C. spartinae DAOMC 251721
C. spartinae RUTPP 3491HT
p8/).977 C. perihumidiphila DAOMC 250581
C. perihumidiphila DAOMC 252161"
C. arundinis CCC 1094
C. arundinis DAOMC 251724=CCC 933€t
C. arundinis CCC 974
C. arundinis CCC 236
C. arundinis CCC 902
C. arundinis CCC 480
TNS-F-60476 Phalaris arundinacea Toyama JA
TNS-F-60478 P. arundinacea Ishikawa JP
TNS-F-60488 P. arundinacea Ishikawa JP
TNS-F-60492 P. arundinacea Tochigi JP
TNS-F-60499 P. arundinacea Gifu JP
TNS-F-60498 Phieum pratense Gifu JP
TNS-F-60500 P. arundinacea Tochigi JP
TNS-F-60506 P. arundinacea Chiba JP"T
TNS-F-60507 P. arundinacea Chiba JP
TNS-F-60512 P. pratense Miyagi JP
TNS-F-60514 P. arundinacea Nagano JP
TNS-F-60516 P. pratense Miyagi JP
DAOM 984879 P. arundinacea Alberta CA
DAOMC 251717=CCC 434 Dactylis sp. Bavaria DEFT
DAOMC 251722=CCC 588 Phieum pretense Panska Skala CZ
TNS-F-60470 Poa annua Ishikawa JP
TNS-F-60472 Poa trivialis Ishikawa JP
TNS-F-60473 Alopecurus aequalis var. amurensis Ishikawa JP
TNS-F-60477 Dactylis glomerata Ishikawa JP
TNS-F-60481 D. glomerata Niigata JP
TNS-F-60482 Poa palustris Niigata JP
TNS-F-60489 Poa acroleuca Ishikawa JP
TNS-F-60490 D. glomerata Chiba JP
TNS-F-60491 D. glomerata Tochigi JP
TNS-F-60493 Calamagrostis epigeios Ishikawa JP
TNS-F-60495 Agrostis gigantea Ishikawa JP
TNS-F-60497 Festuca rubra Ishikawa JP
TNS-F-60503 Polypogon fugax Ishikawa JP
TNS-F-60504 Poa pratensis Ishikawa JP
TNS-F-60505 Poa annua Kanagawa JP
TNS-F-60508 A. aequalis var. amurensis Niigata JP
TNS-F-60518 D. glomerata Miyagi JP
DAOM 984880 F. rubra Alberta CA
» CCC 691 P. arundinacea Dzungar Alatau KZ
CCC 1020 Molinia caerulea Bozi Dar CZ

94/1

40.0

C. bavariensis
wl ¥

77

Fic. 1. Phylogenetic tree based on the concatenated sequence alignments of TEFl-a and RPB2
showing the separation and close relationship of Claviceps humidiphila and C. bavariensis. Reference
sequences are in gray font, showing species names and voucher numbers; 24 taxa in C. sect.
Pussillae are abbreviated to a single branch. The labels of the sequences in clades C. humidiphila and
C. bavariensis include voucher numbers, hosts, locations, and country abbreviations: CA Canada;
CZ Czech Republican; DE Germany; JP Japan; KZ Kazakhstan. Superscripts ET = ex-type, NT =
neotype. Values on branches are bootstrapping values for MP analyses/posterior probability of BI.

78 ... Liu, Tanaka, Kolafik
Taxonomy

Claviceps humidiphila Pazoutova & M. Kolatik,
Fungal Biol. 119(1): 22 (2015) Fic. 2
= Claviceps purpurea var. phalaridis Tanda, J. Agric. Sci. (Tokyo) 24: 84 (1979).

Ho .oryPE (lost)—Japan, Chiba, Chiba, Yukijirushi Farm on Phalaris arundinacea, Aug
1, 1969, Seinosuke Tanda (TUAMH-PA 921).

NEOTYPE (here designated, MBT395372)—Japan, Chiba, Chiba, Inage [= the holotype
locality], 35.6672°N 140.1353°E, on Phalaris arundinacea, June 23, 2017, Eiji Tanaka
(Neotype, TNS-F-60506; ex-type culture MAFF 247310; GenBank LC598958,
LC598987).

SCLEROTIA dark purple (#100336) to black (#05000A), 4-15 x 0.8-1.4 mm,
cylindrical, floating in fresh water. Conrp14 from sclerotial surface hyaline,
aseptate, allantoid, oblong, ovoid, ellipsoid, lunate, 6.0-16.9 x 3.6-5.9 um, L/W
ratio 2.3-2.6.

ASCOSTROMATA light orange (#E2C86C) to purplish red (#E6BOAA). STIPE
filiform, 6-13 mm long, 0.1-0.4 mm wide. CAPITELLUM 0.5-1.9 mm diam.
PERITHECIA obovoid, slightly fusiform to pyriform, 154-314 x 100-157 um.
Asci cylindrical 50-110 x 2.5-5.5 um. Ascosporss filiform, 85-110 um long.

ADDITIONAL SPECIMENS EXAMINED—JAPAN, CuiBA, Sakura, KODAKE, 35.7388°N
140.1661°E, on Phalaris arundinacea, June 23, 2017, Eiji Tanaka (TNS-F-60507; GenBank
LC598959, LC598988), (TNS-F-60525, fruiting bodies derived from TNS-F-60507);
GiFu, Takayama, Hirayu, 36.1917°N 137.5510°E, on P. arundinacea, Aug 14, 2016,
Kazuhito Tanada (TNS-F-60499; GenBank LC598963, LC598992); 36.1914°N
137.5503°E, on Phleum pratense, Aug 14, 2016, Kazuhito Tanada (TNS-F-60498;
GenBank LC598967, LC598996); IsH1kAwa, Nomi, NABETANI, 36.4189°N 136.5575°E,
on P. arundinacea, June 30, 2016, Eiji Tanaka (TNS-F-60488; GenBank LC598961,
LC598990); Uchinada, Kose, 36.6936°N 136.6842°E, on P. arundinacea, June 18,
2016, Eiji Tanaka (TNS-F-60478, MAFF 247302; GenBank LC598960, LC598989),
(TNS-F-60524, fruiting bodies derived from TNS-F-60478); Mryaa1, Ohira, 38.4683°N
140.8833°E, on P. pratense, Aug 10, 2017, Kazuhito Tanada (TNS-F-60512; GenBank
LC598968, LC598997); NAGANO, Nagano, TOGAKUSHI, 36.7711°N 138.0900°E, on P
arundinacea, Aug 1, 2018, Eiji Tanaka (TNS-F-60514; GenBank LC598966, LC598995);
TocuiGl, Tochigi, Fujioka, 36.2711°N 139.6567°E, on P. arundinacea, July 6, 2016,
Kazuhito Tanada (TNS-F-60492, MAFF 247305; GenBank LC598965, LC598994);
Nikko, Okukinu, 36.8681°N 139.3947°E, on P. arundinacea, Aug 15, 2016, Kazuhito
Tanada (TNS-F-60500, MAFF 247310; GenBank LC598964, LC598993); ToyAMa,
Nanto, FUKUMITSU, 36.5636°N 136.8778°E, on P. arundinacea, June 16, 2016, Eiji
Tanaka (TNS-F-60476, MAFF 247301; GenBank LC598962, LC598991).

Hosts—Phalaris, Phleum

ComMMENTS—In the original description of C. purpurea var. phalaridis (Tanda
1979b), the specimens TUAMH-PA 820 and TUAMH-PA 921 were listed as
the first and second specimens examined followed by the word “Holotype.” It is

Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 79

Fic. 2. Claviceps humidiphila: A. Sclerotia on Phalaris arundinacea (TNS-F-60478);
B. Ascostromata (TNS-F-60524) developed from germinated sclerotia (TNS-F-60478);
C. Conidia washed off sclerotia (TNS-F-60478); D. Perithecia semi-embedded in stromata;
E. Asci with thick caps and filiform ascospores (TNS-F-60524). Scale bars: A = 10 mm; B = 2
mm; D = 50 um; C, E= 10 um.

likely that TUAMH-PA 921 was designated as holotype, although PA 820 was
recorded as holotype in Pazoutova & al. (2015), possibly because PA 921 was
erroneously overlooked. Nevertheless, we searched all of Tanda’s research
collections (1979b) using several approaches. Firstly, theabbreviation TUAMH
is not cited in Index Herbariorum (http://sweetgum.nybg.org/science/ih/)
where TUAT is cited as the correct code for Tokyo University of Agriculture
Herbarium (Museum); however, TUAT is currently inactive, and _ its
100,000 specimens were transferred to the National Museum of Nature
and Science, Tokyo (TNS) in 2007. We searched the TNS online database
(http://db.kahaku.go.jp/webmuseum_en) for Claviceps purpurea collected in
Japan; of the 18 specimens located, none had been collected from Chiba or

80 ... Liu, Tanaka, Kolatik

related to TUAMH PA 820 or PA 921. Next, we contacted the herbarium
director and curator (Dr. Masanobu Higuchi and Dr. Tsuyoshi Hosoya), who
recommended contacting Dr. Keiichi Motohashi at Tokyo University directly.
According to a personal communication with Dr. Seinosuke Tanda, Tanda
left all his un-submitted specimens in the laboratory at Tokyo University of
Agriculture, where Dr. Motohashi checked 6000 specimens that had been
collected by Dr. Tanda; 90% represented powdery mildews and the rest
were rust fungi; no Claviceps specimens were found, possibly because other
specimens had been discarded. Based on the collective evidence, it is likely
that the specimens linked to the name C. purpurea var. phalaridis were lost.
Therefore, we hereby designate a new specimen from the type location as
neotype.

According to Tanda (1979b), the sclerotia were of different sizes. One
specimen collected from Asahi-mura, Niigata (PA007), possessed much
shorter sclerotia (2.3-7.7 x 0.6-1.3 mm; n = 215), while others had a wider
size range (2.2-15.4 x 0.5-1.5 mm; n = 599). The number of stromata
developed from sclerotia varied from 1 to 11, for which Tanda (1979b) cited
the dimensions of 0.1-1.6 x 0.2-1.9 mm (capitellum), 2-13 x 0.1-0.9 mm
(stipe), and 4.4-16.9 x 2.4-5.9 um [L/W 2.2-2.6] (conidia; n = 1350). Our
sclerotial, stromatal, and conidial measurements match those cited by Tanda.
In addition, Tanda (1979b) also recorded perithecia at 161-217 x 91-130
um (a slightly narrower range than our data), asci at 74-133 x 1.8-4.2 um
(slightly longer than our data), and ascospores at 72-129 um (similar to our
data).

Claviceps bavariensis M. Kolatik, E. Tanaka & M. Liu, sp. nov. Fig. 3
MB 838352
[= “Claviceps humidiphila” sensu epitype of Pazoutova & al. 2015, non Tanda 1979.]

TypE—Germany, Bavaria, Philippsreut, 48.8564°N 13.6763°E, on Dactylis sp., 1988,
Pazoutova (Holotype, PRM922708 [dried culture on T2 media]; ex-type culture
CCC434; GenBank JX083704, JX083635).

= Claviceps purpurea var. alopecuri Tanda, J. Agric. Sci. (Tokyo) 22: 295 (1977).

= Claviceps purpurea var. dactylidis Tanda, J. Agric. Sci. (Tokyo) 25: 266 (1980).
Differs from C. huimidiphila in amplified TEF1-a region (918 nts) by at least seven sites
(C. humidiphila/C. bavariensis = 271 A/G; 348 A/G; 642 T/C; 675 T/C; 688 C/T; 690
T/C; 860 T/C) that are identical within species.

EryMoLoGcy—referring to the provenance of the type specimen

SCLEROTIA purplish brown (#452D2E), dark brown (#2D1E1E) to black
(#272424), 3-10 x 0.3-1.4(-1.7) mm, ovoid, obclavate, narrow cylindrical,

Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 81

en wg
ie ay}
BN. ! ij

vn

Fic. 3. Claviceps bavariensis. A. Sclerotia on Dactylis glomerata (TNS-F-60477); B. Sclerotia on
Poa annua (TNS-F-60470); C. Sclerotia on Alopecurus aequalis var. amurensis (TNS-F-60473);
D. Sclerotia on Festuca rubra (DAOM 984880); E. Sclerotia on Phalaris arundinacea
(DAOM984879); F. Sclerotia detached from PRM922708 (holotype); G. Sclerotia detached from
DAOM 984880; H. Sclerotia detached from DAOM 984879; I. Longitudinal section near the rind
showing textura prismatica (DAOM 984879); J, K. Ascostromata developed from germinated
sclerotia from D. glomerata (TNS-F-60523), and A. aequalis var. amurensis (TNS-F-60521);
L, M. Cross sections of sclerotia showing the color of inner tissue (DAOM 984879, 984880);
N. Asci showing the thick caps with a central pore (TNS-F-60522); O. Conidiogenesis in
sclerotia (DAOM 984879, 984880); P. Conidia washed off sclerotia (PRM922708, holotype);
Q. Filiform ascospores (TNS-F-60520). Scale bars: A = 5 mm; B, C, F-H, J, K= 2 mm; D, E= 1 mm;
L, M = 200 um; I = 20 um; N-Q = 10 um.

82 ... Liu, Tanaka, Kolatik

curved, or subulate, floating in fresh water, interior white, greyish yellow
(#DOCBAO) to olive brown (#4A423B) from center to margin, or vivid yellow
(#FEE302) to deep yellow (#FFC40C) in center (3A8-4A8). CONIDIOGENOUS
CELL cylindrical, or obovoid, 12-23(-27) x 5-7 um. Conrp1A ovoid,
reniform, cylindrical, (5.5—-)6.1-13.1(-14.3) x (2.3-)2.4-3.9(-4.4) um, L/W
ratio 2.1-2.2.

ASCOSTROMATA pinkish (#FFCOCB) to light brown (#5D4037). STIPE
filiform 5-8 mm long. CaPITELLUM 0.5-1 mm diam. Asci cylindrical,
50-155 x 2.8-5.0 um. Ascosporsgs filiform.

ADDITIONAL SPECIMENS EXAMINED—CANADA, ALBERTA, Beaverlodge, on Festuca
rubra, Aug. 2019, Henry Klein-Gebbinck (DAOM 984880; GenBank MW411019,
MW411021), on Phalaris arundinacea, Aug. 2019, Henry Klein-Gebbinck (DAOM
984879; GenBank MW411018, MW411020). JAPAN, CuHrspa, Sakura, UsuIDEN,
35.7416°N 140.1850°E, on Dactylis glomerata, Jul 4, 2016, Kazuhito Tanada
(TNS-F-60490; MAFF 247304; GenBank LC598969, LC598998); IsH1KAWA, Hakusan,
SHIRAMINE, 36.1150°N 136.6994°E, on Poa acroleuca, Jul 1, 2016, Eiji Tanaka
(TNS-F-60489; GenBank LC598981, LC599010); YORISHINBO, 36.4770°N 136.5647°E,
on Poa annua, Apr 29, 2016, Eiji Tanaka (TNS-F-60470; GenBank LC598977,
LC599006); Kanazawa, MINATO, 36.6450°N 136.6631°E, on Calamagrostis epigejos, Jul
18, 2016, Eiji Tanaka (TNS-F-60493; MAFF 247306; GenBank LC598985, LC599014);
Komatsu, IMAEMACHI, 36.3772°N 136.4492°E, on Poa trivialis, May 9, 2016, Eiji
Tanaka (TNS-F-60472; GenBank LC598979, LC599008); Nomi, SUEDERA, 36.4442°N
136.5081°E, on Poa pratensis, June 9, 2017, Eiji Tanaka (TNS-F-60504; GenBank
LC598976, LC599005); Nonoichi, NAKABAYASHI, 36.5064°N 136.5994°E, on Polypogon
fugax, June 5, 2017, Eiji Tanaka (TNS-F-60503; GenBank LC598986, LC599015);
Suematsu, 36.5103°N 136.5967°E, on Alopecuru. aequalis var. amurensis, May 23, 2016,
Eiji Tanaka (TNS-F-60473; culture MAFF 247299; GenBank LC598974, LC599003);
(TNS-F-60521, fruiting bodies derived from TNS-F-60473); Tsubata, 36.6875°N
136.7589°E, on F. rubra, Jul 24, 2016, Eiji Tanaka (TNS-F-60497; GenBank LC598984,
LC599013); Uchinada, KosE, 36.6758°N 136.6706°E, on D. glomerata, June 18, 2016,
Eiji Tanaka (TNS-F-60477; GenBank LC598970, LC598999); (TNS-F-60523, fruiting
bodies derived from TNS-F-60477); Koyopal, 36.6375°N 136.6300°E, on Agrostis
gigantea, Jul 24, 2016, Eiji Tanaka (TNS-F-60495; GenBank LC598982, LC599011);
IwaTE, Miyako, Kuzakal, 39.6503°N 141.3574°E, on D. glomerata, Aug 7, 2019, Eiji
Tanaka (TNS-F-60518; GenBank LC598973, LC599002); 39.6512°N 141.3574°E,
on Phleum pratense, Aug 7, 2019, Eiji Tanaka (TNS-F-60516; GenBank LC598983,
LC599012); KANAGAWA, Atsugi, NURUMIZU, 35.4347°N 139.3503°E, on PB annua, June
16, 2017, Kazuhito Tanada (TNS-F-60505; GenBank LC598978, LC599007); NiiGATa,
Sanjo, KamiourA, 37.5883°N 139.0124°E, on A. aequalis var. amurensis, July 3,
2017, Kazuhito Tanada (TNS-F-60508; GenBank LC598975, LC599004); Tokamachi,
MATSUNOYAMA, 37.1189°N 138.5908°E, on D. glomerata, June 24, 2016, Eiji Tanaka
(TNS-F-60481; GenBank LC598972, LC599001); 37.0885°N 138.6097°E, on Poa
palustris, June 24, 2016, Eiji Tanaka (TNS-F-60482; GenBank LC598980, LC599009);
TocuiGl, Tochigi, Fujioka, 36.2436°N 136.6614°E, on D. glomerata, Jul 6, 2016,
Kazuhito Tanada (TNS-F-60491; GenBank LC598971, LC599000).

Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 83

Hosts—Alopecurus, Agrostis, _Ammophila, Calamagrostis, Dactylis,
Deschampsia, Festuca, Molinia, Phalaris, Phleum, Phragmites, Poa, Polypogon

COMMENTS—In our phylogenetic tree (Fic. 1), two samples on Alopecurus,
four on Dactylis, six on Poa, and one on Polypogon group in the Claviceps
bavariensis clade, suggesting that Tanda’s two varieties—C. purpurea var.
alopecuri (Tanda 1977), C. purpurea var. dactylidis (Tanda 1981)—and the
samples from Poa and Polypogon (Tanda 1980; Tanda & Kawatani 1980)
might represent this species. In that case, a species name, “C. alopecuri”
stat. nov., could be coined. However we considered the following: 1) the
type specimen of C. purpurea var. alopecuri designated by Tanda (1977)
was lost due to the same reason as C. purpurea var. phalaridis, therefore it
is not possible to verify molecularly that the lost type truly belonged to the
clade of C. bavariensis, which is essential for species delimitation; 2) the
two specimens on Alopecurus in this study were not from Suginami, Tokyo,
Japan (the type location); 3) the name “C. alopecuri” might give the false
impression that the species has a host range limited to Alopecurus, contrary
to the wide host range of the species. Therefore, we avoided elevating the
varietal name to species level, proposing instead a new species name,
C. bavariensis. Normally an epitype can only be replaced via conservation,
but in this case the holotype specimen to which the epitype specimen would
be attached has been lost, and an epitype no longer has nomenclatural status
(Turland & al. 2018, Art. 9.20 Note 8). This allows us to use PRM922708,
the epitype for C. humidiphila Pazoutova & M. Kolarik, as a holotype for
C. bavariensis.

Tanda (1977, 1980, 1981) and Tanda & Kawatani (1980) recorded detailed
sexual and asexual morphological features of samples from Alopecurus,
Dactylis, Poa, and Polypogon, which could represent Claviceps bavariensis. In
general, sclerotia are cylindrical with pointed or blunt ends; stromatal stipes
are reddish brown, cylindrical or filiform, and curved or straight; capitella are
light orange, oblate globose, and verrucose; perithecia are pyriform or ovoid;
asci are narrowly cylindrical with thickened caps; ascospores are colorless and
filiform; and the conidia are 1-celled, hyaline, and cylindrical or ovoid. The
sizes of these characters sclerotia, stromata, perithecia, asci, ascospores and
conidia varied significantly from different host plants (TaBLE 1). Pazoutova
& al. (2015) also provided more detailed morphological description and
comparison with other species for C. bavariensis (as C. humidiphila).
The conidia of C. bavariensis tend to be larger than C. purpurea s.str. and

84 ... Liu, Tanaka, Kolatik

TABLE 1. Claviceps bavariensis: observations
cited in Tanda (1977; 1980; 1981) and Tanda & Kawatani (1980)

A—Host, sclerotial & ascomal dimensions

Host SCLEROTIA (mm) STIPE (mm) CAPITELLA*

height x diameter (mm)
Alopecurus 1.5-5.1 x 0.3-0.9 1.0-4.0 x 0.3-0.5 0.4-0.9 x 0.6-1.3
Dactylis 3.2-11.7 x 0.6-1.7 1.0-14 x 0.1-1.4 0.3-1.5 x 0.3-2.3
Poa 1.5-5.8 x 0.2-1.5 1.0-9.0 x 0.1-0.7 0.4-1.0 x 0.5-1.9
Polypogon 1.2-4.0 x 0.2-1.0 1.0-7.0 x 0.2—-0.6 0.4-1.1 x 0.5-1.4

B—Anatomical dimensions

PERITHECIA ASCI ASCOSPORE CONIDIA CONIDIA
(um) (um) LENGTH (tm) (um) L/W
151-221 x 77-144 75-138 x 2.5-3.7 74-126 5.0-11.3 x 2.6-5.0 2.1-2.3
154-270 x 88-168 81-151 x 1.8-4.2 74-119 4.0-14.8 x 1.8-4.7 1.9-2.8
147-256 x 60-154 77-158 x 1.8-5.3 63-151 3.2-12.6 x 1.6-5.8 2.0-3.0
119-210 x 56-126 91-133 x 2.5-3.9 84-123 5.0-12.8 x 2.4-5.4 —

C. arundinis, although the size ranges can overlap. Although these three species
shared several host genera, population genetic analyses suggested significant
genetic differentiation and limited gene flow among them (Pazoutova & al.
2015). The three species also differ in their ergot alkaloid spectra (Pazoutova
& al. 2000, Negard & al. 2015) and ergochrome pigments (Flieger & al. 2019).
Nevertheless, separation between C. bavariensis and C. humidiphila relies
essentially on molecular evidence (mainly TEF1-a, at least seven constant
nucleotide variance). The host species cited by Pazoutova & al. (2015) included
10 genera mainly from Europe (Belgium, Czech Republic, Germany, France,
Norway, Poland, Turkey), but also from Central Asia (Kazakhstan, one
specimen on P. arundinacea) and North America (USA, one specimen on
Ammophila breviligulata). The present study expanded sample locations and
host range by adding Canada (on Festuca, Phalaris) and Japan (on Agrostis
gigantea, Alopecurus aequalis var. amurensis, Calamagrostis epigejos, Dactylis
glomerata, Festuca rubra, Poa acroleuca, P. annua, P. palustris, P. pratensis,
P. trivialis, Polypogon fugax).

Claviceps bavariensis sp. nov. & C. humidiphila neotypified ... 85

Acknowledgments

We are grateful to MSc Kazuhito Tanada for providing many ergot samples
from various regions in Japan and Dr. Henry Klein-Gebbinck for samples from
Alberta (Canada). We also thank Drs. Masanobu Higuchi, Tsuyoshi Hosoya,
Keiichi Motohashi and Seinosuke Tanda for their efforts in searching for type
specimens; Dr. Scott Redhead (DAOM, Agriculture and Agri-Food Canada,
Ottawa, ON Canada) for nomenclature advice and pre-submission manuscript
review; and Dr. Joey Tanney (DAVP, Pacific Forestry Centre, Victoria, BC, Canada)
for pre-submission review. We thank Dr. Shaun Pennycook for the nomenclature
review and the meticulous edits that have significantly improved the clarity and
efficiency of the presentation. This work was supported by the Japan Society for the
Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI)
(grant number 16K07238); and Agriculture and Agri-Food Canada STB fungal and
bacterial biosystematics J-002272.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 89-94
https://doi.org/10.5248/137.89

Passalora golaghati comb. nov. from India

GARGEE SINGH’, SANJAY YADAV’,
RAGHVENDRA SINGH?, SHAMBHU KUMAR?

‘Department of Botany, DDU Gorakhpur University,
Gorakhpur, U.P, India 273009

? Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University,
Varanasi, U.P, India 221005

° Forest Pathology Department, KSCSTE-Kerala Forest Research Institute,
Peechi, Thrissur, Kerala, India 680653

" CORRESPONDENCE TO: drsinghtaxon@gmail.com, singhr.bot@bhu.ac.in

ABSTRACT—The hyphomycete Cercospora golaghati is recombined as Passalora golaghati
based on critical re-examinations of original type material and a fresh topotypic collection
and comparison with closely related species of cercosporoid taxa.

Key worps—foliicolous, anamorph, Mycosphaerellaceae, nomenclature, taxonomy

Introduction

The significant characteristic that separates the two cercosporoid genera
Cercospora Fresen. ex Fuckel and Passalora Fr. is colouration of conidia.
When Crous & Braun (2003) emended the circumscription of Passalora
based on ITS and 5.8S rDNA sequence analyses, they observed that the
formation of single or catenate conidia is not a stable feature for diagnosis
at the generic level in cercosporoid hyphomycetes (Crous & al. 2001).
Their contributions have confirmed that presence or absence of thickened
conidiogenous loci and pigmentation in conidiophores and conidia are
important features of taxonomic relevance (Crous & al. 2009, 2013; Videira
& al. 2017). During the last decade a large number of cercosporoid fungi
have been recombined in the genus Passalora (Crous & Braun 2003; Braun

90 ... Singh & al.

& al. 2013, 2014, 2015a,b, 2016), particularly from India (Kamal 2010).
Recently, the addition of several novel taxa of foliicolous cercosporoid fungi
described from India (Kumar & Singh 2015a,b, 2016; Awasthi & al. 2016;
Singh & al. 2011, 2012, 2013) suggests that cercosporoid diversity is still
insufficiently known in this region.

A new collection from the type locality of Cercospora golaghati (Saikia
& Sarbhoy 1980) confirmed that this species is characterized by thickened
scars and coloured conidiophores and conidia, indicating that it should be
transferred to Passalora (Crous & Braun 2003).

Materials & methods

The holotype [conserved in the Herbarium, Division of Mycology & Plant
Pathology, Indian Agricultural Research Institute, New Delhi, India (HCIO)] and a
fresh specimen collected from the same host species in the same locality [conserved
in the Mycological Herbarium of the Gorakhpur University, Gorakhpur, UP, India
(MH-GPU)] were critically analyzed, and slides from infection spots were mounted
in distilled water, lactophenol and cotton-blue mixture. Observation of microscopic
characters was recorded under an Olympus CH20i-TR compound microscope to
understand the exact morphology of the fungus. Measurements of 30 conidia, hila,
conidiophores, and conidiogenous cells were recorded with the help of stage and
ocular micrometers, and Lucida drawings were also made. The morphology of the
fungus was compared with closely related cercosporoid taxa with the help of current
literature.

Taxonomy

Passalora golaghati (Saikia & A.K. Sarbhoy) Gargee Singh, Sanj. Yadav,
Raghv. Singh & Sh. Kumar, comb. nov. Figs 1-4

MB 835579
= Cercospora golaghati Saikia & A.K. Sarbhoy, Curr. Sci. 49: 830. 1980, as “golaghatii”.

INFECTION SPOTS amphigenous, brown to dark blackish brown, initially
circulartosubcircular, later spreading over the leafsurface to become irregular,
3-15 mm in diam. CoLonigs hypophyllous and effuse. MycELrum internal.
STROMATA present, epidermal to subepidermal, pseudoparenchymatous,
brown to dark brown, 30-32 x 18-19 um in diam. CONIDIOPHORES
fasciculate, macronematous, brown to dark brown, unbranched, cylindrical,
erect to procumbent, straight to flexuous, geniculate, smooth, thick-
walled, 2—7-septate, (62—)80-102(-152) x 3-5 um. CONIDIOGENOUS CELLS
polyblastic, integrated, terminal, apex often slightly wider than at the base,
20-40 x 3-5 um, loci thickened and darkened, 2-2.5 um. Conip1a solitary,
acropleurogenous, obclavate-cylindrical, simple, dry, straight to slightly

Passalora golaghati comb. nov. (India) ... 91

Fics 1-4. Passalora golaghati (holotype, HCIO 32660).
1. Symptoms of infection; 2. Stroma; 3. Conidiophores; 4. Conidia.

92 ... Singh & al.

curved, thin-walled, smooth, brown to mid brown, 3-6-septate, tip acute
to sub-obtuse, base obconico-truncate, (35-)50-60(-75) x 2.5-6 um, hilum
protuberant, thickened and darkened, 2-2.5 um.

SPECIMENS EXAMINED: INDIA, Assam, Golaghat, Salikihat, on living leaves of
Dioscorea alata L. (Dioscoreaceae), 21 November 1977, leg. U.N. Saikia (HCIO 32660,
holotype); 26.5239°N 93.9623°E, on living leaves of Dioscorea alata, December 2019, leg.
Gargee Singh (MH-GPU 1, topotype).

Discussion

A literature review revealed that seven Passalora taxa have been
reported on Dioscoreaceae: P. dioscoreae (Ellis & G. Martin) U. Braun &
Crous, P. dioscoreae-nipponicae Y.L. Guo, P. dioscoreae-subcalvae Y.L. Guo,
P. dioscoreicola Y.L. Guo, P. dioscoreigena U. Braun & Crous, P. tranzschelii
(Vassiljevsky) U. Braun & Crous var. tranzschelii, and P._ tranzschelii
var. chinensis Y.L. Guo (Braun & al. 2014, Crous & Braun 2003;
Guo 2001, 2011). Passalora dioscoreae has been subsequently recombined
in Distocercosporaster by Videira & al. (2017). Passalora golaghati differs
distinctly from all the remaining Passalora species in its conidiophore and
conidial sizes. Morphological comparisons of all Passalora species reported
on Dioscoreaceae are presented in TABLE 1.

Key to Passalora spp. reported on Dioscoreaceae

DesConidial leriot ie 75 phi’. se Pan. otteela-nongdanaotedasscrtlancs aden scar a da sarte tends, sels. dee 2
ESCO eial Pei ot ly A Ty hy aha ect obs oct tey Iafemattee Bhanchey inc epaorabay bhsuably beube weed ial ate 3
2. Conidiophores 35-175 x 4-6 um, conidia 10-55 x 7.5-10 um,

Pe2eseptate or dihioes shines cGine Ling iat PGA whut eae 8 NGUoS P. dioscoreicola
2. Conidiophores 80-102 x 3-5 um, conidia 50-60 x 2.5-6 um,

S=G-SEPlate: 2 Sis isos we Seb vG Seo eG s 6. Bye 8 we Ben vG ed So Rea P. golaghati
3-Conidiophorelenctins5 Oui, <4 sei «2 ven cam 8 cGy epee any eee eee es 4
8. Conidie plore: le tect i 50) quite 1k 5 hae ee Prt PS ee Md Re el 5
4. Conidiogenous loci 1-2 um diam., conidia 15-90 x 2-5 um,

W=S=septate”, sh. eg gis ea gistn +a site tga pera 4s bier ds P. tranzschelii var. tranzschelii
4, Conidiogenous loci 1.5-3 um diam., conidia 30-125 x 4-7 um,

SS VOAseptateny cin. egies gd ge eddie ya ceerds Herd he P. tranzschelii var. chinensis
5s Comidiaphorelene ty AT OOM: i..5-3 dreseu-g dros setog gras sekeg rhs pebog echt pebon xis feds (9 pete g tt pede 6
SeGonidiophore lemethy SOO I ses otatse-o + eatery 4 tarsal ata cy aaa awe ote P. dioscoreigena
6. Conidiophores 10-70 x 4-7 um, conidia 30-125 x 4-6.5 um,

S—PO=SE PLA LES toh su sea tsa testes a cet bed cp sto Srsghiuova aeblade asses P. dioscoreae-nipponicae

6. Conidiophores 25-65 x 4.5-6.5 um, conidia 30-85 x 4.5-6.5 um,
DA~GUSEPLALG Eh Ore stance ries veks Sere aseantes tame cic ldes Russab Veh gcc Taneg P. dioscoreae-subcalvae

Passalora golaghati comb. nov. (India) ... 93

TABLE 1. Comparative morphology of Passalora taxa reported on Dioscoreaceae.

CONIDIA

TAXON CONIDIOPHORE SIZE (um)

SEPTATION SIZE (um)
P. dioscoreae-nipponicae 10-70 x 4-7 3-10 30-125 x 4-6.5
P. dioscoreae-subcalvae 25-65 x 4.5-6.5 2-6 30-85 x 4.5-6.5
P. dioscoreicola 35-175 x 4-6 1-2 10-55 x 7.5-10
P. dioscoreigena 40-120 x 5-8.5 1-6 25-110 x 4.5-6.5
P. golaghati 80-102 x 3-5 3-6 50-60 x 2.5-6
P. tranzschelii var. tranzschelii 5-50 x 3-7 0-3 15-90 x 2-5
P. tranzschelii var. chinensis 5-50 x 3-7 3-10 30-125 x 4-7

Acknowledgments

Sincere thanks are due to Curator of the HCIO (New Delhi) for making it possible
to examine the holotype collection of Cercospora golaghati. We express our deep
gratitude to Dr. R.E Castafeda-Ruiz (INIFAT Alejandro de Humboldt, Habana,
Cuba) and Dr. Flavia Rodrigues Barbosa (Universidade Federal de Mato Grosso,
Brazil) for presubmission critical review. We express our deep appreciation to Prof.
Dr. Kamal (Emeritus Scientist, DST) for his valuable suggestions and kind help. We
are also thankful to the Head of the Department of Botany of D.D.U. Gorakhpur
University, Gorakhpur, U.P., for providing necessary facilities.

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https://doi.org/10.1016/j.simyco.2017.09.003

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 95-107
https://doi.org/10.5248/137.95

Stigmatomyces aff. limnophorae on dipteran hosts
in Peninsular Malaysia

NATASHA AZMI NurR-ALIAH’, JINGYU LIU’,
Nurut AZMIERA', CHONG CHIN HEO??”

' Department of Medical Microbiology and Parasitology,
Faculty of Medicine, Universiti Teknologi MARA Selangor Branch,
Sungai Buloh Campus, 47000 Sungai Buloh, Selangor, Malaysia

? Department of Botany and Plant Pathology, College of Agriculture,
Purdue University, West Lafayette IN 47907 U.S.A.

° Institute for Pathology, Laboratory and Forensic Medicine (I-PPerForM),
Universiti Teknologi MARA Selangor Branch,
Sungai Buloh Campus, 47000 Sungai Buloh, Selangor, Malaysia

* CORRESPONDENCE TO: chin@uitm.edu.my

ABSTRACT—Flies parasitized by Laboulbeniales, captured in Selangor state, were examined
and identified as Boettcherisca javanica, Boettcherisca sp., and Hypopygiopsis violacea.
The fungus was identified as Stigmatomyces aff. limnophorae based on morphology and
phylogenetic analysis of sequences of the partial nuclear small and large subunit ribosomal
RNA genes. This fungus represents a new record for Malaysia; and these are the first reports
of Boettcherisca and Hypopygiopsis as hosts for any laboulbenialean species.

Key worps—Calliphoridae, Laboulbeniomycetes, Sarcophagidae, southeastern Asia.

Introduction

Fungi in Laboulbeniales (Ascomycota: Laboulbeniomycetes) are
ectoparasitic fungi that are obligatorily associated with arthropods as
ectoparasites (Haelewaters & al. 2012, Melo & Melo 2019, Blackwell & al.
2020). These fungi are characterized by the presence of a three-dimensional
thallus (plural: thalli), instead of hyphae with mycelial growth like many other
fungi (Blackwell & al. 2020). Representatives of three arthropod subphyla

96 ... Nur Aliah & al.

(Chelicerata, Myriapoda, Hexapoda) and various insect orders including
Coleoptera, Diptera, Hemiptera, and Hymenoptera are known as hosts for
these fungi (reviewed in Haelewaters & al. 2021). In the past 40 years, only
two species Laboulbeniales have been reported in Malaysia: Laboulbenia
admirabilis, found on the body of an unidentified Spaniocelyphus (Diptera)
in Pahang state (Lee & Majewski 1986), and Diphymyces sabahensis, on
three Ptomaphaginus spp. (Coleoptera) in Sabah state (Haelewaters & al.
2014).

Stigmatomyces sensu lato is a large, paraphyletic genus of 176 species
on dipteran hosts (Haelewaters & al. 2020a; Species Fungorum 2020).
Species within this heterogeneous assemblage (sensu lato) are parasites on
hosts in many different families—including Anthomyiidae, Calliphoridae,
Chamaemyiidae, Diopsidae, Drosophilidae, Ephydridae, Fanniidae,
Muscidae, Nycteribiidae, Sarcophagidae, Sphaeroceridae, and Streblidae
(Thaxter 1901, 1905, 1917; Rossi 1998; Hyde & al. 2019; Haelewaters &
al. 2018, 2020a). Thus far, five species of Stigmatomyces sensu stricto have
been reported in Malaysia: S. dacinus, S. limosinoides, S. tortimasculus, and
S. venezuelae in Malaysian Borneo (Thaxter 1915, 1918); and S. neurochaetae
in Peninsular Malaysia (Sugiyama & Majewski 1985; Rossi & Weir 2007).
Note that Thaxter (1915) reported S. stilici from Malaysian Borneo and
Sugiyama & Majewski (1985) reported S. orientalis from Peninsular
Malaysia—both associated with staphylinid beetles (Coleoptera:
Staphylinidae) and both species later recombined in Zeugandromyces.

Here, we provide the first records of Stigmatomyces aff. limnophorae from
Malaysia. Our material was studied based on morphological characters and
sequence data. The parasitized fly genera are for the first time reported in
the literature as hosts for Laboulbeniales.

Material & methods

Collection & identification of flies

An entomological survey was conducted in September 2019 in the state of
Selangor, Peninsular Malaysia, to investigate the biodiversity of carrion flies. Chicken
liver (200 g, 2d old) was used as bait and flies were collected using sweep nets at two
different forests in the town of Rawang. Collected adult flies were then placed in a
cloth-lid jar and brought back to the Parasitology Laboratory, Institute for Medical
Molecular Biotechnology, Universiti Teknologi MARA (UiTM) in Sungai Buloh.
The flies were incubated at -4 °C for 15 minutes, after which they were pinned
and dried at room temperature. During microscopic examination for species
determination, we observed four fly specimens (out of >100 observed) with thalli of

Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 97

Laboulbeniales. These specimens were carefully examined and photographed using
an Olympus SZ51 stereomicroscope equipped with a digital camera and CellD
Imaging Software. The parasitized adult flies were identified using Kurahashi & al.
(1997) and Kurahashi & Samerjai (2018).

Microscopic study of Laboulbeniales

Parasitized flies were shipped to Purdue University for microscopic study of
the Laboulbeniales (by J.L.). Thalli were taken from the host fly using a BioQuip
#1208SA entomological pin dipped in Hoyer’s medium (30 g arabic gum, 200 g
chloral hydrate, 16 ml glycerol, 50 ml ddH20). Thalli were mounted in Amann’s
medium applying a double coverslip technique using Solakryl BMX as outlined in
Liu & al. (2020). Microscope mounts were viewed at 200-400x using an Olympus
BH2 bright field compound microscope. Line and stipple drawings were made with
PITT artist pens based on photomicrographs taken with an Olympus SC30 camera
and cellSens 1.18 imaging software. Permanent slides are deposited at PUL (Kriebel
Herbarium) under numbers PUL F25943-F25950.

DNA extraction, PCR amplification, sequencing

DNA was extracted from 2-4 thalli of Stigmatomyces using the REPLI-g Single
Cell Kit with modifications by Haelewaters & al. (2019). The nuclear ribosomal
RNA small (SSU) and (LSU) large subunits were amplified using primer pairs
NSL1/NSL2 for SSU (Haelewaters & al. 2015), and LROR/LR5 and LIC24R/LR5
for LSU (Vilgalys & Hester 1990, Hopple 1994, Miadlikowska & Lutzoni 2000).
The DNA was amplified using an Eppendorf pro S Mastercycler in 25 uL volumes
containing 12.5 ul 2x MyTaq Mix (Bioline, Swedesboro, New Jersey), 9.5 uL ddH20,
1.0 uL forward and reverse primer, and 1.0 uL DNA. Cycling conditions—for SSU:
initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 30
sec, annealing at 55 °C for 45 sec, extension at 72 °C for 45 sec; and final extension
at 72 °C for 1 min and for LSU: initial denaturation at 94 °C for 5 min; 35 cycles
of denaturation at 94 °C for 30 sec, annealing at 50 °C for 45 sec, extension at 72
°C for 1 min; and final extension at 72 °C for 7 min. The PCR amplicons were
sent to Genewiz (South Plainfield, New Jersey) for purification and sequencing.
Raw sequence reads were assembled and edited in Gene Codes Sequencher 5.2.3.
Sequences were deposited at the National Center for Biotechnology Information
(NCBI) GenBank database; accession numbers MT341792—-M1T341794 (SSU)
and MT341789-MT341791 (LSU). These sequences were then BLAST searched
against NCBI's nucleotide collection to establish a rough relationship with existing
sequences.

Sequence alignments & phylogenetic analysis

SSU and LSU sequences of Stigmatomyces species representing the same
clade (clade IV sensu Haelewaters & al. 2020a) were downloaded from GenBank
(https://www.ncbi.nlm.nih.gov/genbank/). Gloeandromyces dickii was selected as
outgroup. Details for all isolates are presented in TABLE 1. Sequences of both regions

98 ... Nur Aliah & al.

TABLE 1. Isolates and sequences used in phylogenetic analysis.
SPECIES ISOLATE Country SSU LSU REFERENCE

Gloeandromyces dickii D.Haelew. Panama MG958011 MH040582 Haelewaters & al. 2018

1323b
Stigmatomyces borealis AW-979 USA JN835186 — A. Weir
(unpublished)
S. chamaemyiae D.Haelew. Portugal MH040564 — Haelewaters & al. 2018
1137a
D.Haelew. Portugal MH040565 — Haelewaters & al. 2018
1137c
S. limnophorae AW-785 USA AF407576 — Weir & Blackwell 2001b
S. aff. limnophorae D.Haelew. Malaysia MT341792 MT341789 This study
1802c
D.Haelew. Malaysia MT341793 MT341790 This study
1802d
D.Haelew. Malaysia MT341794 MT341791 This study
1820e
S. protrudens AW-793 USA AF298232 AF298234 Weir & Blackwell 42001a
S. rugosus — — AF431759 — Weir & Hughes 2002
D.Haelew. Portugal MH040563 — Haelewaters & al. 2018
1138a

were aligned with MUSCLE (Edgar 2004) on the Cipres Science Gateway web portal
(Miller & al. 2010). For both datasets, the appropriate nucleotide substitution model
was selected by considering the corrected Akaike Information Criterion (AICc) using
ModelFinder Plus (Kalyaanamoorthy & al. 2017). Models selected were HKY+F+I
(SSU, -InL = 2420.547) and TIM2+F+I (LSU, -InL = 1837.675). SSU and LSU aligned
datasets were combined using MEGA7 (Kumar & al. 2016). A Maximum likelihood
analysis of the concatenated two-locus dataset was performed using IQ-TREE
(Nguyen & al. 2015) with partitioned models (Chernomor & al. 2016) and ultrafast
bootstrapping with 1000 replicates (Hoang & al. 2018). The best-scoring tree was
visualized in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/ software/figtree/) and edited in
Adobe Illustrator 24.1.1.

Host identification

The infected insect hosts (n = 4) belonged to two families, Calliphoridae
(n = 1) and Sarcophagidae (n = 3). The calliphorid fly was identified as
Hypopygiopsis violacea (Fic. 1). Two sarcophagid flies were identified as
Boettcherisca javanica and the third as Boettcherisca sp. (Fics 2, 3). All four
specimens bore thalli at their abdominal segments (TABLE 2).

Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 99

Fic. 1. Adult fly of Hypopygiopsis violacea (Diptera: Calliphoridae): A. Habitus of H. violacea at 0.8x
magnification. B. The silver white facial tomentum. C. Stigmatomyces aff. limnophorae thalli on 5th
tergite (black arrow). D. Stigmatomyces aff. limnophorae thalli on sternites 3 and 4 (black arrows).
Scale bars: A, B= 2mm; C, D=1 mm.

tom ity Tea
Fic. 2. Adult fly of Boettcherisca sp. (Diptera: Sarcophagidae): A. Heavy infection with Stigmatomyces
aff. limnophorae at abdominal tergites 4 and 5 (black arrow); B. Close-up view of the parasitized
tergites 4 and 5 at 2.5x magnification. Scale bars: A = 2 mm; B = 1 mm.

Fig. 3. Adult fly of Boettcherisca javanica (Diptera: Sarcophagidae): A. Stigmatomyces aff.
limnophorae thalli on abdominal sternites 4 and 5 (black arrow); B. Tuft of S. (aff.) limnophorae
thalli on abdominal sternites 4 and 5 at 3.2x magnification. Scale bars” A = 2 mm; B= 1 mm.

100 ... Nur Aliah & al.

TABLE 2. Host specimens examined and position of thalli on the host body.

DIPTERAN HOST SPECIES FAMILY LOCATION OF THALLI

Hypopygiopsis violacea Calliphoridae Tergite 5; sternites 3, 4 (Fic.
Q 1C, D)

Boettcherisca sp. Sarcophagidae Abdominal tergites 3, 4
Q (Fic. 2)

Boettcherisca javanica Sarcophagidae Abdominal sternites 4, 5
rou (Fia. 3)

Taxonomy

Stigmatomyces aff. limnophorae Thaxt.,
Proc. Am. Acad. Arts Sci. 36: 400. 1901. Fia. 4
Thallus hyaline, dark amber brown that gradually attenuates as the cell
wall thickens; 647 um long from foot to perithecial tip. Cell I] <2 times
longer than cell I, striate on the cell surface. Cell III longer than wide,
rounded externally, not protruding abruptly below basal cell of appendage.
Appendage free, slender, elongated, about as long as the perithecial venter;
distal portion distinctly curved; bearing a single or multiple antheridia.
Antheridia, short, broad, slightly recurved. Perithecium amber brown, 220
x 64 um; venter relatively small, ellipsoid, subsymmetrical, with wall cells
powdered by darker maculation, spirally twisted, separated by corresponding
number of well-defined longitudinal ridges, somewhat oblique; neck 96 x 16
um, abruptly distinguished from venter.

MATERIAL EXAMINED/SEQUENCED—PENINSULAR MALAYSIA: SELANGOR,
Gombak District, RAWANG, forested area, 3.281°N 101.261°F, 4 m a.s.l., ex. chicken
liver, 11 Sep. 2019, leg. N.A. Nur Aliah & N. Azmiera, on Q Boettcherisca sp. D.
Haelew. 1796 [host label] (slides PUL F25943, PUL F25944, PUL F25945); 3.296°N
101.611°E, 69 ma.s.l., ex. chicken liver, 7 Sep. 2019, leg. N.A. Nur Aliah & N. Azmiera,
on & Boettcherisca javanica Lopes, D. Haelew. 1801 [host label] (slides PUL F25947,
PUL F25948); on CO B. javanica, D. Haelew. 1802 [host label] (slides PUL F25949,
PUL F25950) isolate 1802c [4 mature thalli] GenBank MT341792, MT341789; isolate
1802d [3 mature thalli] GenBank MT341793, MT341790; isolate 1802e [2 perithecia]
GenBank MT341794, MT341791); 3.296°N 101.611°E, 101 ma.s.l., ex. chicken liver, 7
Sep. 2019, leg. N.A. Nur Aliah & N. Azmiera, on Q Hypopygiopsis violacea (Macquart),
D. Haelew. 1800 [host label] (slide PUL F25946).

Phylogenetic results
All three newly generated SSU sequences share highest similarity (99.62%)
with Stigmatomyces chamaemyiae (GenBank MH040565), followed by

Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 10.1

Fig. 4. Stigmatomyces aff. limnophorae: A [PUL F25945], B [PUL F25948]. Details of primary
appendage; C [PUL F25950]. Detail of spirally twisted perithecium; D [PUL F25950].
Receptacular cells I and II with longitudinally striped ornamentation; E [PUL F25950]. Habitus
of mature thallus; F. Stipple drawing. Scale bars: A, B = 10 um; C, D = 20 um; E = 50 um;
F= 100 um.

S. rugosus (MH040563) with 98.39-98.54% similarity (TaBLE 1). Based on
morphology, we thought the fungus might represent S. limnophorae, but
that species was not among the BLAST results despite the availability of an
SSU sequence in GenBank (AF407576; isolate AW-785). Comparison of our
Malaysian SSU sequences with S. limnophorae AW-785, however, shows a
99.08-99.36% similarity. All newly generated LSU sequences are most closely
related to Gloeandromyces nycteribiidarum (MH040566) with 85.78% similarity
(TABLE 1). The highest percentage similarity (87.30%) with any Stigmatomyces
species is with S. protrudens (AF298234), but with only over a 42% query cover.

The three Malaysian isolates group together in a maximum-supported clade
that also includes S. limnophorae AW-785 and two S. chamaemyiae isolates
from Portugal. The branch length among the Malaysian isolates and between
the USA isolate AW-785 is very short. As a result, we believe the Malaysian
fungus may be identified as S. aff. limnophorae.

Discussion
Fungal thalli were removed from all four fly specimens and subsequently
identified as Stigmatomyces aft. limnophorae based on morphological (Fic. 4)

102 ... Nur Aliah & al.

Gloeandromyces dickii D. Haelew. 1323b

Stigmatomyces protrudens AW-793
Stigmatomyces borealis AW-979

Stigmatomyces rugosus

Stigmatomyces rugosus D. Haclew. 1138a
Stigmatomyces cf. limnophorae D. Haclew. 1802¢.
Stigmatomyces cf. limnophorae D: Haclew.1802d

Stigmatomyces\limnophorae AW-185)

Stigmatomyces chamaemyiae D. Haelew. 1137a

0.02
Stigmatomyces chamaemyiae D. Haelew. 1137¢

Fig. 5. Phylogeny of Stigmatomyces isolates reconstructed from a combined SSU-LSU rDNA
dataset, with Gloeandromyces dickii as outgroup. Shown is the best-scoring tree (-InL =
4263.622) as a result of maximum likelihood inference performed with IQ-TREE. For each
node, the ML bootstrap (if >60) is presented at the branch leading to that node. Stigmatomyces
aff. limnophorae is highlighted in grayscale.

and phylogenetic (Fic. 5) analyses. The global distribution of S. limnophorae
is presented in TABLE 3. Stigmatomyces limnophorae was described from a
Limnophora fly (Muscidae) in California, USA. Isolate AW-785 originated
from a Muscidae gen. sp. indet. collected in Louisiana, USA (Weir & Blackwell
2001b). This isolate will be regarded as representing the species until fungal
sequences are obtained from a muscid fly identified to genus-level collected in
the type locality of Berkeley, California. Morphologically our material resembles
typical S. limnophorae morphology (Thaxter 1901, 1908), but our phylogenetic
placement of the Malaysian isolates sister to S. chamaemyiae + S. limnophorae
AW-785, suggests that they might represent another species closely related to
S. limnophorae. Since the vast majority of thalli were heavily damaged, we were
unable to describe the material accurately based on all morphological features.

Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 103

TABLE 3. World distribution of Stigmatomyces limnophorae,
with host species and reference of first report.

CONTINENt COUNTRY Host (FAMILY) FIRST REPORT
North & Central America Cuba Limnophora arcuata (Muscidae) Krejzova & Weiser 1968
Grenada Anthomyiidae gen. sp. indet. Thaxter 1917
Guatemala = Limnophora sp. (Muscidae) Thaxter 1917
Jamaica Leucomelina sp. (Muscidae) Thaxter 1917
Mexico Onesia sp. (Calliphoridae) Thaxter 1917
USA Limnophora sp. (Muscidae) [type] Thaxter 1901
Anthomyiidae gen. sp. indet. Thaxter 1917
South America Bolivia Limnophora sp. (Muscidae) Rossi 1998
Brazil Limnophora sp. (Muscidae) Bergonzo & al. 2004
Venezuela Sarcophaga sp. (Sarcophagidae) Thaxter 1905
Europe Portugal Limnophora obsignata (Muscidae) Rossi & al. 2013
Africa Cameroon Anthomyiidae gen. sp. indet. Thaxter 1917
Canary Limnophora obsignata (Muscidae) Rossi & al. 2013
Islands
Kenya Rhyncomya forcipata (Rhiniidae) Rossi & al. 2013
Morocco Limnophora obsignata (Muscidae) Rossi & al. 2013
Sierra Leone _ Lispe desjardinsii (Muscidae) Rossi & Leonardi 2018
Uganda Fainia albitarsis (Rhiniidae) Rossi & al. 2013
Asia Indonesia Lucilia dux (Calliphoridae) Thaxter 1917
Philippines — Lucilia dux (Calliphoridae) Thaxter 1917
Thailand Heliographa ceylanica (Muscidae) Rossi & al. 2013
Israel Limnophora quaterna (Muscidae) Rossi & al. 2013
Saudi Isomyia terminata (Rhiniidae) Rossi & al. 2013
Arabia
Limnophora quaterna (Muscidae) Rossi & al. 2013
Taiwan Sumatria flava (Rhiniidae) Rossi & al. 2013
Turkey Dasyphora albofasciata (Muscidae) Rossi & al. 2013
Australasia Australia Calliphora augur (Calliphoridae) Rossi & al. 2013

As a result, for the time being, we refer to the species as S. aff. limnophorae.
Efforts will be ongoing to sample flies using chicken liver and other baits and to
collect additional Stigmatomyces-infected fly specimens.

104 ... Nur Aliah & al.

Stigmatomyces limnophorae was discovered by Thaxter (1901) as a parasite
of Limnophora sp. [misspelt as “Limnophorus”] (Muscidae) in California, USA.
The fungus appears to be a very widespread and plurivorous species and has
been reported on all continents except Antarctica from hosts in different
dipteran families including Anthomyiidae, Calliphoridae, Muscidae, Rhiniidae,
and Sarcophagidae (TABLE 3). Despite its wide distribution, the fungus has not
yet been reported in Malaysia. Stigmatomyces limnophorae has, however, been
reported in neighbouring countries—Indonesia, the Philippines, Thailand—
on different dipteran hosts such as Chrysomya megacephala [as “Lucilia dux”]
(Calliphoridae) and Heliographa ceylanica (Muscidae) (Thaxter 1917, Rossi &
al. 2013). Here we report the occurrence of S. aff. limnophorae in Malaysia on
different hosts in Calliphoridae and Sarcophagidae. While it is not uncommon
for S. limnophorae to parasitize flies in either of these families (TABLE 3), the
Malaysian hosts are in genera that have not previously been observed with
thalli of Laboulbeniales.

Thalli of Stigmatomyces aff. limnophorae were always observed at the
abdominal segments, which might indicate the original infection site; however,
our observations are based on only three host specimens. Limited data are
available on Laboulbeniales. For example, the impact of infection on their hosts
is still poorly studied (Nalepa & Weir 2007, Riddick 2010, Bathori & al. 2017,
Haelewaters & al. 2020b). Further exploration of the diversity of ectoparasitic
fungi is required to gain a complete picture of their distribution and interactions
with insect hosts.

In summary, we report two new hosts for Stigmatomyces limnophorae,
a complex species able to infect wide number of species within different
genera. This could be caused by evolution and adaption of the fungi for
survival in different genera on various continents. As our sample was
identified morphologically as S. limnophorae but phylogenetically as sister to
S. chamaemyiae + S. limnophorae AW-785 (suggesting a different species),
we could postulate that hybridization is occurring between these two species,
thereby explaining adaptation of these fungi to different host species and
within wide range of genera. Additional research is required to support this
hypothesis.

Acknowledgments

We thank the Institute of Medical Molecular Biotechnology (IMMB), Universiti
Teknologi MARA, for providing laboratory facilities. Special thanks to Dr. Matthew
T. Kasson (West Virginia University, USA) for comments during our early research
and to Dr. Danny Haelewaters (Purdue University, USA) for help with phylogenetic

Stigmatomyces aff. limnophorae on dipteran hosts (Malaysia) ... 105

analyses. Dr. Haelewaters and Dr. Walter P. Pfliegler (University of Debrecen,
Hungary) are thanked for expert presubmission review.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 109-121
https://doi.org/10.5248/137.109

Pluteus variabilicolor and Volvopluteus earlei,
new records for Pakistan

JUNAID KHAN’, HASSAN SHER’, AIMAN IZHAR?’,
MUHAMMAD HAQNAWAZ?’, ABDUL NASIR KHALID?

"Center for Plant Sciences and Biodiversity, University of Swat,
Mingora, Pakistan

? Institute of Botany, University of the Punjab, Quaid-e-Azam campus,
Lahore, Pakistan

*CORRESPONDENCE TO: junaid.botany@gmail.com

Axsstract—Pluteus variabilicolor and Volvopluteus earlei are reported as new records
for the Pakistani funga, with the former representing a new record for South Asia and the
latter, a second report from Asia. Morphological descriptions are accompanied by colored
photographs of basidiomata and microscopic structures. Identity of both species is confirmed
by ITS sequence analyses. Comparison with previous descriptions and data on their
distributions are briefly discussed.

Key worps—Agaricales, phylogeny, Pluteaceae, P. sect. Hispidoderma, taxonomy

Introduction

Pluteaceae Kotl. & Pouzar (Agaricales) is a family of saprophytic fungi
widely distributed in major parts of the world (Kirk & al. 2008). Molecular
reassessment of the family (Justo & al. 2011a) supports two genera: Pluteus
Fr. and Volvopluteus Vizzini & al.

Pluteus, the larger genus, comprises >300 species (Kirk & al. 2008, He
& al. 2019), distributed worldwide especially in boreal to tropical forested
regions (Menolli & al. 2014). Pluteus species are primarily lignicolous and
grow on partially or well-rotted angiosperm and gymnosperm wood (Orton
1986, Singer 1986, Vellinga 1990). The presence of free lamellae, pinkish

110... Khan & al.

spore-print, inverse hymenophoral trama, and inamyloid basidiospores and
lack of universal veil are some key Pluteus characters (Vellinga & Schreurs
1985, Orton 1986, Singer 1986, Vellinga 1990, Heilmann-Clausen 2012).
Only eight Pluteus species have been reported from Pakistan: P ephebeus
(Fr.) Gillet (as P murinus Bres.), P escharites (Berk. & Broome) Sacc.,
P. fusconigricans (Berk. & Broome) Sacc., P. leoninus (Schaeff.) P. Kumm.,
P. palumbinus (Berk.) Sacc., P. pellitus (Pers.) P. Kumm., P. petasatus (Fr.)
Gillet, and P pulverulentus Murrill (Ahmad 1980, Iqbal & Khalid 1996,
Ahmad & al. 1997).

Volvopluteus, typified by V. gloiocephalus (DC.) Vizzini & al., was erected
to accommodate some species formerly placed in Volvariella (Justo & al.
2011a). The genus is distinguished from Pluteus and Volvariella by a gelatinous
pileipellis that is an ixocutis with relatively narrow hyphae (average <15 um
diam.) and average basidiospore length >11 um (Justo & al. 2011a). Only
one Volvopluteus species, V. gloiocephalus, has been reported from Pakistan
(Ahmad 1980 and Iqbal & Khalid 1996, as Volvariella speciosa (Fr.) Singer).

This paper, part of a larger project dealing with the exploration of
macrofungi in northern Khyber Pakhtunkhwa reports two pluteaceous
species as new records for the funga of Pakistan: Pluteus variabilicolor and
Volvopluteus earlei.

Materials and methods

Collection & morphological Studies

Specimens were collected during macrofungal surveys conducted between 2014
and 2019 in different parts of Pakistan. The specimens of P. variabilicolor were
collected in village Ingaro Dherai, district Swat, Khyber Pakhtunkhwa province.
Those of V. earlei were collected in the same locality and in the Sheikupura and
Muzaffargarh districts of Punjab province. Basidiomata were dug out to the extreme
base (to obtain some wood with specimens of Pluteus and volva with those of
Volvopluteus) with a sharp knife. The specimens were photographed in their natural
habitat and important macro-morphological characters were noted when fresh.
Colors were coded according to soil color chart of Munsell (1975). Terminology
for macroscopic and microscopic features follows Vellinga (1988). The specimens
were dried in sun and deposited in the herbarium of the University of Swat, Kanju
Township, Pakistan (SWAT) and the herbarium of the University of the Punjab,
Lahore, Pakistan (LAH).

Microscopic descriptions are based on free-hand sections from dried specimens
mounted both in distilled water and in aqueous potassium hydroxide solution (5%
w/v), with aqueous Congo red added for contrast. Basidiospore measurements
are based on 60 elements randomly selected from all the available basidiomata;

Pluteus & Volvopluteus spp. new to Pakistan ... 111

x presents the mean length and width of the basidiospores. The abbreviation Q refers
to the length : width (L/W) ratio, with Q = the average length : width ratio.

DNA extraction, PCR amplification, sequencing

Genomic DNA from dried herbarium specimens was extracted using modified
CTAB method of Gardes & Bruns (1993). The internal transcribed spacer region
(ITS = ITS1-5.8S-ITS2) was amplified using the primer pair ITSIF (Gardes &
Bruns 1993) and ITS4 (White & al. 1990). Polymerase chain reactions (PCR) were
performed in 25 uL volume aliquots. Amplification involved initial 4 minutes
denaturation at 94 °C, 40 cycles of 1 minute at 94 °C, 1 min at 55 °C, 1 min at 72 °C,
and a final extension of 10 minutes at 72 °C (Naseer & al. 2020). PCR products were
visualized in a UV illuminator loaded in 1% agarose gel added with 3 uL ethidium
bromide. Purification and sequencing of the PCR amplicons were carried out at
Beijing Genomic Institute (BGI) Hong Kong.

Sequence alignment & phylogenetic analysis

The forward and reverse reads were assembled into a consensus sequence using
BioEdit software v. 7.2.5 (Hall 1999). The consensus sequences were used for a
GenBank search, using the NCBI Basic Local Alignment Search Tool (BLAST)
(https://blast.ncbi.nlm.nih.gov/Blast.cgi). Matching sequences, especially those
published in Justo & al. (2011b) and Lezzi & al. (2014), were downloaded for further
phylogenetic analysis. Separate datasets were used for Pluteus and Volvopluteus.

Multiple sequences were aligned using online Multiple Alignment using Fast
Fourier Transform (MAFFT) algorithm at European Bioinformatics Institute
website (https://www.ebi.ac.uk/Tools/msa/mafft/). The phylogenies were inferred
by maximum likelihood (ML) analysis using model selection for the best DNA
analysis in MEGA6 software (Tamura & al. 2013). Jukes-Cantor model Models with
the lowest BIC scores (Bayesian Information Criterion) was considered to describe
the substitution pattern the best. Non-uniformity of evolutionary rates among sites
were modeled using a discrete gamma distribution (+G) with 5 rate categories
and assuming that a certain fraction of sites is evolutionarily invariable (+1).
The phylogenetic analyses included 1000 bootstrap replicates.

Phylogenetic results

The final Pluteus ITS dataset comprised 37 sequences representing P sect.
Hispidoderma Fayod including the two sequences from our new Pakistani
specimens. Pluteus diettrichii Bres. (HM562143) of P sect. Celluloderma
Fayod was used as outgroup following Lezzi & al. (2014). Maximum
likelihood analysis convincingly clustered the Pakistani collections
with other sequences of P. variabilicolor in the Leoninus clade of P. sect.
Hispidoderma with a 100% bootstrap value (Fic. 1).

The ITS consensus sequences from our three new Pakistani Volvopluteus
collections matched 99-99.5% with multiple V. earlei sequences (HM562205,

112... Khan & al.

HM562139 Pluteus aff. leoninus TNSF11908
MGS544913 Pluteus roseipes
HM562215 Pluteus leoninus DrewH
HM562071 Pluteus leoninus Halling6546
t- HM562187 Pluteus leoninus SF17
KC147679 Pluteus roseipes UC 1861249
KC147681 Pluteus roseipes UC 1861251 P. leoninus complex
99) HM562188 Pluteus aff. leoninus | SF19
HMS562190 Pluteus aff. leoninus | SF21
HM562077 Pluteus leoninus Josserand s.n.
MG544910 Pluteus leoninus
98 79) HM562045 Pluteus leoninus AJ212
MG544911 Pluteus leoninus
KP192913 Pluteus variabilicolor
MT351154 Pluteus variabilicolor (SWAT001367)
KP192914 Pluteus variabilicolor
MG544909 Pluteus variabilicolor
MG544908 Pluteus variabilicolor
1'FJ774077 Pluteus aurantiorugosus P. variabilicolor
86 HM562099 Pluteus castroae
HMS562092 Pluteus castroae
MT351155 Pluteus variabilicolor (LAH31413)
bi KP192911 Pluteus variabilicolor
KP192912 Pluteus variabilicolor
JN603206 Pluteus chrysaegis K13587
94] )HM562142 Pluteus conizatus var. africanus | P. chrysaegis
88. MG968799 Pluteus chrysaegis
KF692077 Pluteus pantherinus
100. HM562089 Pluteus pantherinus
100) HM562048 Pluteus granulatus
HM562048 Pluteus granulatus

= 100} MH059512 Pluteus umbrosoides Teapdhveatdey

69

52

| P. pantherinus

P. granulatus

KX216348 Pluteus umbrosoides
69 KX216343 Pluteus umbrosus
to0| | KX216347 Pluteus umbrosus
63] }HM562069 Pluteus granularis
96'HM562189 Pluteus granularis
HM562143 Pluteus diettrichii | outgroup

P. umbrosus/granularis

0.02

Fic. 1: Maximum likelihood phylogram of Pluteus variabilicolor and related species inferred
from ITS data. Bootstrap values 250% are presented above nodes. Newly generated sequences of
P. variabilicolor are presented in bold.

HM562205, HM246496, HM246497, HM246498, HM246499, MK204987,
MK204989) in GenBank. The final Volvopluteus dataset comprises 19
sequences including the three sequences from the new Pakistani specimens
and from Pluteus heteromarginatus Justo (HM562058) and P longistriatus
(Peck) Peck. (HM562082), the outgroup cited in Justo & al. (2011b).
Sequences of the Pakistani collections clustered with other sequences of V.
earlei, confirming their identity (Fic. 2).

Pluteus & Volvopluteus spp. new to Pakistan ... 113

6g | HM562204 Volvopluteus earlei MA22816

HM246499 Volvopluteus earlei TO HG1973
HMS562205 Volvopluteus earlei Mamet7

MK204987 Volvopluteus earlei

MK204989 Volvopluteus earlei

HM246496 Volvariella cookei TO AV133

HM246498 Volvariella media TO HG2001

93 || MT353644 Volvopluteus earlei SWAT001366
MW362280 Volvopluteus earlei LAH35715
OM761893 Volvopluteus earlei LAH37018
HM246497 Volvopluteus earlei TO AV 134

MK944280 Volvopluteus sp. JPL 2019

100' MK 944281 Volvopluteus sp. JPL 2019 (GDGM73195)
MK616346 Volvopluteus gloiocephalus
MK616345 Volvopluteus gloiocephalus
NR 119878 Volvopluteus asiaticus

NR 119876 Volvopluteus michiganensis

HMS562058 Pluteus heteromarginatus

100 HM562082 Pluteus longistriatus

61

99

=
0.02

Fic. 2: Maximum likelihood phylogram of Volvopluteus earlei and related species inferred from
ITS data. Bootstrap values 250% are presented above nodes. Newly generated sequences of V. earlei
are presented in bold.

Taxonomy

Pluteus variabilicolor Babos,
Ann. Hist.-Nat. Mus. Natl. Hung. 70: 93 (1978) Fics 3a-c, 4
PiLEus 50-70 mm diam., convex to plano-convex with a central umbo,
light orange yellow (10YR 9/8) to dark yellow (2.5Y 9/12), disc darker
(2.5Y 7/12); surface smooth, glabrous to lightly velvety, margin striate or
not; context concolorous with the pileus just beneath the cuticle, otherwise
cream colored, unchanging upon cutting, thin, 2-3 mm at the disc, <1 mm
at the margin. LAMELLAE free, close, sub-ventricose (<6 mm at the center);

114... Khan & al.

white to pale pink (7.5YR 9/4) when young, pink (7.5R 9/4) when mature;
lamellar edge even to slightly undulate; lamellulae present, short to long
extending beyond the middle of the lamellae. StrpE 50-80 x 5-7 mm, central,
cylindrical to very slightly thickening downward; pinkish (7.5YR 8/4);
longitudinally striate in mature specimens, fistulose; context concolorous
with the exterior or light yellowish brown (10YR 6/4), unchanging upon
cutting.

BASIDIOSPORES (5.1-)5.5-6.3(-6.8) x (4.4-)4.8-5.8(-5.9) um, x = 5.9
x 5.1 um, Q = 1.1-1.19(-1.2), Q. = 1.1, mostly sub-globose, rarely ovoid
to broadly ellipsoid; smooth, thick-walled (2-3 um thick), mostly with
one central guttula, rarely multi-guttulate. Basidia 20-25 x 6.0-7.0 um;
cylindrical to broadly oblong, with 4 sterigmata. CHEILOCysTIDIA 55-90 x
20-30 um; fusiform with a short appendix at the tip to lageniform.
PLEUROCYSTIDIA 60-120 x 20-40 um, narrowly utriform to utriform,
rare. PILEIPELLIS a hymeniderm composed of clavate to rounded terminal
elements, 50-200 x 20-40 um, without any yellow intracellular pigment.
STIPITIPELLIS a cutis composed of narrow branched hyphae, 3.0-5.0 um
diam., with rare claviform caulocystidia, 25-50 x 15-30 um, usually in
clusters extending along the stipe length. CLAMP CONNECTIONS absent.

MATERIALS EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Ingaro
Dherai village, 1000 m. a.s.l, on decomposing stump of Populus nigra L., 7 August
2014, Junaid Khan ING-32 (SWAT001367; GenBank MT351154); Ingaro Dherai
village, 1010 m. a.s.l., on decomposing stump of Populus nigra, 27 July 2015, Junaid
Khan ING-1507 (LAH31413; GenBank MT351155).
COMMENTARY—Pluteus variabilicolor, originally described from Hungary,
is characterized by its pileus color varying from yellowish orange to chrome-
yellow, pileipellis with morphologically variable elements (a mixture of
spheropedunculate-vesiculose and elongated + cystidioid elements), the
presence of caulocystidia, and growth on decaying sawdust (Babos 1978).
Our description fits well with previous descriptions (Lohmeyer & al. 1994,
Lanconelli & al. 1998, Migliozzi 2011, Lezzi & al. 2014, Kaygusuz & al.
2019).

Lezzi & al. (2014) cite P variabilicolor as widespread but not common.
Currently the species has been reported from numerous central and eastern
European countries—Hungary (Babos 1978), Austria (Lohmeyer & al.
1994), Italy (Lanconelli & al. 1998, Migliozzi 2011), Germany (Ludwig
2007), central Russia (as P castri, Justo & al. 2011b), Romania (Béres 2012),
Slovenia (Jogan & al. 2012), and Moldova (Lezzi & al. 2014). From Asia,
the species has been reported from Japan (as P. castri, Justo & al. 2011b),

Pluteus & Volvopluteus spp. new to Pakistan ... 115

Fic. 3: Field photographs of basidiomata. Pluteus variabilicolor: a—b. (SWAT001367);
c. (LAH31413). Volvopluteus earlei: d & f (SWAT001366); e. (LAH37018); g & h. (LAH35715).
Scale bars = 10 mm.

and from Turkey and South Korea (Kaygusuz & al. 2019). Here we report
P. variabilicolor from Pakistan and South Asia for the first time.

Pluteus variabilicolor grows primarily on sawdust deposits and on
branches and rotten wood of Fagaceae, mainly Quercus spp. (Migliozzi
2011, Lezzi & al. 2014). Both Pakistani collections, however, were collected
from decaying stumps of Populus sp., which agrees with Russian reports
of collections growing on Populus tremula stumps of (Justo & al. 2011b).

116... Khan & al.

Fig. 4: Pluteus variabilicolor (SWAT001367): a. Basidiospores; b. Basidia; c-f. Cheilocystidia;
g-i. Pleurocystidia; j, k. Pileipellis elements; |. Stipitipellis elements. Scale bars: a, b = 5 um;
c-i = 20 um; j-l = 30 um.

We suggest that P variabilicolor was introduced into Pakistan with the
introduction of Populus nigra.

Volvopluteus earlei (Murrill) Vizzini, Contu & Justo,
Fungal Biology 115(1): 15 (2011) Fics 3d-h, 5
PiLeus 20-40 mm diam.; hemispherical to obtusely conical when young,
applanate to plano-concave on maturity; white to greyish white with a
greyish disc when young, in maturity white to moderate whitish pink

Pluteus & Volvopluteus spp. new to Pakistan ... 117

Fic. 5: Volvopluteus earlei (LAH35715): a. Basidiospores; b. Basidia; c. Cheilocystidia;
d. Pleurocystidia; e. Stipitipellis; f. Pileipellis elements. Scale bars = 10 um.

(2.5YR 7/4) with reddish pink margins (10R 6/8); surface moist and viscid
in young specimens, dry and dull when mature, glabrous; margin striate,
striation short (<1/4 of pileus radius); context white, thin, 1-2 mm thick at
the disc, <1 mm elsewhere. LAMELLAE free, broad (4-6 mm at the center),
sub-crowded to close; white to cream-colored when young, pink (7.5YR 8/8)
when mature; lamellulae present. Stipe 70-100 x 4-7 mm; central, tapering
upward from slightly flattened base; surface smooth; white to cream colored,
turning slightly pinkish (7.5 YR 9/4) upon maturity and handling; volva

118 ... Khan & al.

present, small (<8 mm high), membranous, saccate, 2-3 lobed; white to pale
gray.

BasIDIOSPORES (11.0—)11.5-14.5(-15.0) x (7.0-)7.5-9.6(-10.0) um, x =
12.8 x 9.1 um, Q = (1.2-)1.3-1.6(-1.7), Q. = 1.4; ellipsoid to ovoid, smooth,
thick-walled (<0.5 um), mostly with one large and many small guttulae.
Basip1A 30-40 x 10.0-12.0 um, 4-spored, cylindrical to broadly clavate.
CHEILOCYSTIDIA 35-60 x 8.0-15.0 um, fusiform to clavate mucronate.
PLEUROCYSTIDIA 80-150 x 15-20 um, cylindrical to narrowly conical.
PILEIPELLIS ixocutis, composed of cylindrical and scattered obovoid
to broadly cylindrical and rarely branched terminal elements; 30-35 x
14.0-18.0 um. STIPITIPELLIS hyphae 7-10 um diam., + parallel, caulocystidia
cylindrical to narrowly clavate, 40-55 x 8-11 um. CLAMP CONNECTIONS
absent.

MATERIAL EXAMINED: PAKISTAN, KHYBER PAKHTUNKHWA, Swat district, Ingaro

Dherai village, 1000 m. a.s.l., among grasses in humus rich wet soil in the paddy fields,

27 June 2016, Junaid Khan ING-1501 (SWAT001366; GenBank MT353644); PUNJAB,

Sheikhupura district, 236 m. a.s.l., solitary on muddy rich soil of a graveyard, 16

July 2017, Aiman Izhar SKP03 (LAH35715; GenBank MW362280); Muzaffargarh

district, surrounding head Taunsa barrage, 65 m. a.s.l., in scattered groups on humus

rich soil, 13 August 2019, Muhammad Haqnawaz TM04 (LAH37018; GenBank

OM761893).
COMMENTARY— Volvopluteus earlei, originally described from Cuba (Murrill
1911), is characterized by its dry to sub-viscid relatively small (25-45 mm)
white pileus, narrowly cylindrical stipe with a small white basal volva,
rare or absent broadly clavate pleurocystidia, rostrate cheilocystidia, and
large (averaging >12.5 tm long) basidiospores (Shaffer 1957, Desjardin &
Hemmes 2001, Contu 2007, Justo & al. 2011a). Murrill (1911) described
only basidiospores in his description; other microscopical details were later
provided by Coker (1947) and Shaffer (1957). Descriptions from different
parts of the world (Pathak 1975, Heinemann 1975, Desjardin & Hemmes
2001, Contu 2007, Vizzini & Contu 2010, Justo & al. 201 1a) cite highly diverse
cystidial forms for V. earlei. For instance, no cystidial forms were reported
for the African (Pathak 1975) and Italian (Vizzini & Contu 2010) collections
of V. earlei [= Volvariella acystidiata N.C. Pathak]. Elsewhere, Justo &
Castro (2010) reported only cheilocystidia in the Spanish collection, while
Shaffer (1957) report both cheilocystidia and pleurocystidia for the North
American collections We observed both cheilocystidia and pleurocystidia
in the Pakistani collection, which agrees with descriptions of the American
collections (Shaffer 1957, Desjardin & Hemmes 2001).

Pluteus & Volvopluteus spp. new to Pakistan ... 119

Volvopluteus earlei is known from the U.S.A (Murrill 1911; Coker
1947; Desjardin & Hemmes 2001), Mexico (Vazquez & al. 1989), Africa
(Heinemann 1975, as Volvariella acystidiata), Sardinia (Contu 2007, as
Volvariella earlei (Murrill) Shaffer), Spain (Justo & Castro 2010), and
India (Amandeep & al. 2015; Atri & al. 1996, as Volvariella earlei). This is
the first report of this species from Pakistan and the second record from
Asia.

Acknowledgments

We are grateful to Dr. Alfredo Justo (Curator of Botany and Mycology, New
Brunswick Museum, Saint John, Canada) and Dr. Nelson Menolli Jr., (Professor,
Federal Institute of Education, Science and Technology, Sao Paulo, Brazil) for
reviewing this manuscript.

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https://doi.org/10.1016/b978-0-12-372180-8.50042-1

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 123-134
https://doi.org/10.5248/137.123

New Turkish records of
Hebeloma excedens and H. geminatum,
and confirmation of H. celatum

AYTEN Dizxrricr’, AYSENUR KALMER’, ISMAIL ACAR”

"Department of Molecular Biology and Genetics, Van Yiiziincti Yil University,
65080, Van, Turkey

? Department of Organic Agriculture, Baskale Vocational High School,
Van Yiiztincti Yl University, 65080, Van, Turkey

* CORRESPONDENCE TO: iacar2011@gmail.com

ABSTRACT—Hebeloma excedens and H. geminatum are reported as new records and
H. celatum is confirmed for the Turkish mycota, on the basis of morphological and
phylogenetic data. Morphological descriptions, illustrations, and comparisons with closely
related taxa are provided.

Key worps—Agaricales, Basidiomycota, Hymenogastraceae, ITS, phylogeny

Introduction

Hebeloma (Fr.) P. Kumm. (Hymenogastraceae, Agaricales) is a genus of
ectomycorrhizal and mostly poisonous basidiomycetes primarily distributed
in temperate, boreal, arctic, and alpine habitats (Vesterholt 2005, Beker & al.
2016, Cripps & al. 2019). The genus is characterized by a mostly two coloured
convex to plano-convex pileus, dull brown lamellae, presence of a veil, and
morphologically distinctive cheilocystidia (Beker & al. 2016).

Identification of Hebeloma at the species level is considerably challenged
by morphological similarities and infrequent or transient characters
(Eberhardt & al. 2012). Therefore, species determination and classification
and their phylogenetic relationships within the genus have come to rely
upon DNA genomic sequences such as the nuclear ribosomal DNA internal

124 ... Dizkirici, Kalmer, Acar

transcribed spacer (nrDNA ITS) region. One advantage of selecting the ITS
region for analysis is the many Hebeloma sequences available in GenBank.
Moreover, the availability of universal primers, high rate of PCR success, and
superior resolution at infrageneric levels are other important advantages of
the ITS region (Aanen & al. 2000, 2001; Vesterholt & al. 2014; Eberhardt &
al. 2015a,b).

Twenty-eight Hebeloma species have been recorded from Turkey (Sesli &
Denchev 2014; Giingor & al. 2015; Sesli & al. 2015, 2018; Solak & al. 2015;
Dogan & Kurt 2016; Dizkirici & al. 2019). Beker & al. (2016) investigated
seven Turkish specimens (representing six species, including H. celatum
and H. subtortum) using molecular data but did not provide morphological
details; subsequently Dizkirici & al. (2019) provided detailed descriptions of
Turkish H. subtortum. Hebeloma celatum, which has been widely reported
from Europe, known from Turkey only by a single 2008 collection from
Adana province (Grilli & al. 2015). Here, we provide full descriptions and
phylogenetic results for Hebeloma excedens, Hebeloma geminatum, and
Hebeloma celatum.

Hebeloma excedens is placed in H. sect. Hebeloma, characterized by the
visible cortina seen in young basidiomes and cheilocystidia that are always
lageniform or ventricose; H. geminatum belongs to H. sect. Denudata,
characterized by cheilocystidia that are primarily clavate-stipitate or clavate-
ventricose and basidiospores that are weakly dextrinoid; and H. celatum
is placed in H. sect. Velutipes, characterized by a velutinate stipe, clavate
cheilocystidia, strongly dextrinoid basidiospores, and absence of cortina
(Vesterholt 2005, Beker & al. 2016, Grilli & al. 2015).

In the present study, we used morphological and ITS sequence analyses to
identify H. excedens and H. geminatum as new Turkish records and confirm
the existing Turkish record of H. celatum.

Materials & methods

Taxon sampling & morphological studies

Fresh basidiomata of H. excedens, H. geminatum, and H. celatum were collected
during 2017-18 fieldwork in south-eastern Turkey. Collected specimens were
photographed in situ using a Canon (EOS 60D) camera equipped with a Tokina
100 mm macro lens. Macromorphological characters were determined based on
field notes and colour photographs of fresh fruiting bodies. Dried specimens were
examined microscopically after sectioning and rehydration following procedures
in Vesterholt (2005) and Beker & al. (2016). Structures were observed using a Leica
DM500 research microscope and measured with the Leica Application Suite v.3.4.0.

Hebeloma celatum, H. excedens, H. geminatum in Turkey... 125

The samples were dried and deposited as voucher specimens in the Fungarium of
Van Yiiztincti Yil University, Van, Turkey (VANF). Abbreviations include: x = average
length x width and Q = length/width ratio of basidiospores calculated based on the
number (n) of spores measured. At least 100 spores, 30 basidia, and cheilocystidia
from four samples were measured in distilled water and Melzer’s reagent for each
specimen. Additional potentially diagnostic characters were abbreviated according
to Vesterholt (2005) with “D” indicating the degree of basidiospore dextrinoidity in
Melzer’s, “P” the degree of loosening of the basidiospore perisporium in Melzer'’s,
and “O” the degree of ornamentation on the basidiospore surface.

DNA isolation, amplification, sequencing

Genomic DNA was extracted and purified directly from dried basidiomata using a
modified CTAB protocol (Doyle & Doyle 1987). The purity and quantity of extracted
DNA were determined by using a NanoDrop2000c UV-Vis Spectrophotometer and
0.8% agarose gel electrophoresis. Isolated stock DNA was stored at -20°C prior for
further studies.

Two different samples were amplified for each Hebeloma species to increase
reliability of the sequences. DNA was amplified in 25 pl volume mixtures containing
genomic DNA (10 ng/ul), 10X PCR Buffer, MgCl, (25 mM), dNTP mixture (10
mM), the primer pair (10 uM), Taq polymerase (5u/ul) and sterile water. The primer
pair N-ncl8S10 5’AGGAGAAGTCGTAACAAG3’ / C26A 5’GTTTCTTTTCCTCCGCT3’
(Wen & Zimmer 1996) was used to amplify the ITS1-5.8S-ITS2 region. Amplicons
were checked in 1% TAE agarose gel stained with Gelred dye. The PCR products
were sequenced with forward and reverse primers in an ABI 3730XL automated
sequencer. Sequences generated in the current study were submitted to GenBank.

Sequence alignment and phylogenetic analysis

All sequence chromatograms were opened using Finch TV (http://www.geospiza.
com/finchtv/) and checked for reading errors, using the Q-Quality value to assess the
accuracy of each base. BLAST analysis was performed using the UNITE (http://unite.
ut.ee/) and NCBI (http://www.ncbi.nlm.nih.gov/) databases. Following a preliminary
BLAST search that supported our newly amplified sequences in Hebeloma, we
prepared a combined dataset containing sequences scoring the greatest number of
BLAST hits.

Sixty-three sequences representing different Hebeloma sections were downloaded
from the database and aligned with the newly amplified sequences. The final
alignment was trimmed, with the data retained for further phylogenetic analyses.
Galerina pruinatipes (AJ585510) and G. pseudocamerina (AJ585508) were chosen as
outgroup.

The nucleotide evolutionary model for phylogenetic analyses was determined
using MEGA 6.0 (Tamura & al. 2013), with the model with the lowest BIC (Bayesian
Information Criterion) score selected. MrBayes v.3.2.6 (Ronquist & al. 2012) was
employed for Bayesian phylogenetic analysis using the Markov chain Monte Carlo
(MCMC) method (Yang & Rannala 1997) under K2P model. Two independent runs

126 ... Dizkirici, Kalmer, Acar

with 4 chains each were run for 2 million generations, with trees sampled every
1000 generations. Analyses continued until the average standard deviation of split
frequencies was <0.01. A Bayesian inference 50% majority rule tree and posterior
probability values were estimated from the samples after discarding the first 25% of
sampled trees and viewed with Figtree (Rambaut 2010).

Taxonomy

Fic 1. Hebeloma excedens [VANF1051]: a. Basidiomata; b. Basidiospores in distilled water;
c. Basidiospores in Melzer’s reagent; d. Basidia; e. Cheilocystidia; f. Pileipellis. Scale bars:
a-e = 20 um; f = 50 um.

Hebeloma excedens (Peck) Sacc.,
Syll. Fung. 5: 806 (1887) FIG. 1

PiLeus 15-25 mm, convex then almost flat and slightly umbonate, darker
in the center (cocoa brown, yellow brown, or cream brown when young; later
darkening), pale brown on most of the pileus, lighter at margin and with/
without white velar remnants on the edges. CorTINA present. LAMELLAE
sinuate, subdecurrent, whitish cream when young, then pale brown, wider
and eroded in age. STIPE 30-60 x 3-5 mm, cylindrical, equal, slightly curved,
pale cream to light brown, pruinose above cortina zone, lower parts dirty and
pale brown, with zones of brown fibrils.

Hebeloma celatum, H. excedens, H. geminatum in Turkey ... 127

BasiD1Iospores 7.8-11(-12) x 4.1-6.3 um, x = 9.8 x 5.2 um, (n = 40;
Q = 1.8-1.9), light yellow-yellow brown, elliptical, slightly ovoid, slightly
roughened (O1), non- to slightly dextrinoid (D0; D1), perispore not loosening
(PO). Basip1a 19.7-31 x 7-9.8 um, clavate, 4-spored. PLEUROCYSTIDIA
absent. CHEILOCYSTIDIA 35-58 x 5-7 x 4.6-6.8 x 5.6-11 tm, ventricose,
cylindrical apex, swollen at the base, rarely fully cylindrical, sometimes
septate. PILEIPELLIS an exocutis, <45 um thick, slightly brownish, encrusted.

SPECIMEN EXAMINED—TURKEY, BiTLis, Van-Bitlis roadway, under Populus sp.,
38.3625°N 42.7614°E, 1952 m asl, 15.05.2018, Acar 1051 (VANF1051; GenBank
MW544166, MW544167).

Hebeloma geminatum Beker, Vesterh. & U. Eberh.,
Persoonia 35: 122 (2015) FIG. 2

PiLeus 25-110 mm, usually convex, occasionally umbonate or flattened,
edges generally smooth, slightly incurved or wrinkled when young, when
young sticky or moist, usually uniformly coloured (occasionally bi-coloured),
cream to beige or (sometimes) light yellow, whitish toward margin. CORTINA
absent. LAMELLAE close, adnate to emarginate, notched, finely serrate at
margin, droplets on margin when young or in humid conditions, color light
pinkish-grey. Stipe 30-100 x 4-12 mm, flocculose, cylindrical to clavate
(slightly swollen toward base), whitish.

BASIDIOSPORES 10-11.8 x 5.5-6.5 um, x = 7.6 x 11.5 (n = 40; Q = 1.7-1.8),
amygdaloid, non- to very slightly dextrinoid (D0,D1), ornamentation distinct

Fic 2. Hebeloma geminatum [VANF1116]: a. Basidiomata; b. Basidiospores in distilled water;
c. Basidiospores in Melzer’s reagent; d. Basidia; e. Cheilocystidia in Melzer’s reagent;
f. Pileipellis. Scale bars: a—e = 20 um; f = 50 um.

128 ... Dizkirici, Kalmer, Acar

verrucose (02,03), perispore loosening (P0,P2). BAsrp1a 31 x 8 um, 4-spored
(rarely 2-spored). PLEUROCYSTIDIA absent. CHEILOCYSTIDIA 50-85 x 7.2-11 x
4-5.6 x 3.3-4.5 um, clavate-stipitate, spathulate, sometimes clavate-lageniform
or sometimes capitate/bifurcate, septate, sinuate. PILEIPELLIS an ixocutis; with
a medium thick epicutis 110-180 um thick, tramal hyphae beneath subcutis
cylindrical, ellipsoid, sausage-shaped and inflating from septa <15 um diam.

SPECIMEN EXAMINED—TURKEY, BINGOL, Elmali village, under Populus sp., 39.0256°N
40.7339°E, 1234 m, asl 20.05.2017, Acar 1116 (VANF1116; GenBank MW544168,
MW544169).

Fic 3. Hebeloma celatum [VANF1132]: a. Basidiomata; b. Basidiospores in distilled water;
c. Basidiospores in Melzer’s reagent; d. Basidia; e. Cheilocystidia; f. Pileipellis. Scale bars:
a-e = 20 um; f= 50 um.

Hebeloma celatum Grilli, U. Eberh. & Beker,
Mycol. Progr. 15(5): 23. 2015 [“2016”] FIG. 3

PiLeus 30-70 mm, convex, occasionally wrinkled, margin involute when
young, sometimes smooth or wavy when mature, glutinous when damp,
spotting variable, mono- or bi-coloured, cream, clay-buff, ochraceous or
yellowish brown. Cortina absent. LAMELLAE adnexed to emarginate, white
fimbriate margin usually present. STrPE 30-70 x 6-10(12) mm, white to light
cream, surface with white powdery granules, fibrils, or floccules; cylindrical,
clavate with bulbous base, rarely tapering, stuffed when young, later hollow.

BASIDIOSPORES 9-15.2 x 5.5-8.5 um, x = 11.3 x 5.2 um, (n = 40;
Q = 1.6-1.8), amygdaloid, limoniform, yellow, yellowish brown, light yellow,
reddish yellow to brown, apiculus distinct, guttulate, ornamentation distinct

Hebeloma celatum, H. excedens, H. geminatum in Turkey ... 129

verrucose (O2-O4), strongly dextrinoid (D2-D4), perispore loosening
(PO-P2). Basip1a 25-38 x 7-9.8 um, cylindrical or clavate, 4-spored, rarely
2-spored. PLEUROCYSTIDIA absent. CHEILOCYSTIDIA 32-75 x 4-10 x 4-6.8 x
4-—11.2 um; cylindrical, clavate, clavate-lageniform, or ventricose, sometimes
subcapitate, septate. PILEIPELLIS an ixocutis; epicutis 70-95 um thick,
tramal hyphae beneath subcutis angular, ellipsoid, sausage-shaped, inflating
from septa <18 um diam. CLAMP CONNECTIONS occasionally present on
cheilocystidia.

SPECIMEN EXAMINED— TURKEY, HAKKARI, Semdinli, DERYA VILLAGE, under Quercus
sp., 37.3383°N 44.5322°E, 1616 m, 15.10.2018, Acar 1132 (VANF1132; GenBank
MW544170, MW544171).

Phylogenetic results

Phylogenetic analyses were performed on the ITS dataset comprising
71 sequences, of which six were derived from our Turkish samples and two
represented the outgroup. The 5’ and 3’ends were trimmed from the ~680
bp sequence alignment and of the 631 bp included in the final analysis, 130
were variable and 87 parsimony informative. Of the variable sites, the ITS1
possessed 72, the 5.8S gene only one, and the ITS2 57. BLAST and UNITE
results of ITS sequences indicated 99% identity value for each sample.

Our Bayesian phylogenetic tree (Fic. 4) outlines the phylogenetic
relationships and taxonomic positions among the species selected. Our
Hebeloma excedens samples (VANF1051A,B) clustered with H. excedens,
H. mesophaeum, and H. subtortum in H. sect. Hebeloma with strong support
(PP = 0.97). The two H. excedens sequences grouped together with a PP = 1
value, expected because no nucleotide variation was observed between them.
Differences between our and the database sequences were noted at ITS1 base
150 (C to T) and ITS2 base 530 T to C).

Our Hebeloma geminatum (VANF1116A, B) samples grouped closely
with the database representatives in H. sect. Denudata (PP = 1), with
H. geminatum closely related to H. alpinum (J. Favre) Bruchet, H. aanenii
Beker & al., H. crustuliniforme (Bull.) Quél., H. eburneum Malencon,
H. salicicola Beker & al., and H. minus Bruchet (Fic. 4). Hebeloma geminatum
cannot be easily separated from its sister species based on ITS sequence data
alone; here morphological characters are helpful. Eberhardt & al. (2015a)
observed that a specimen almost certainly represents H. geminatum when
the average width of the cheilocystidial apex exceeds 9 um. The average
cheilocystidial width in our material was 8-9 um but its range extending to
11 um supports its identification as H. geminatum.

130 ... Dizkirici, Kalmer, Acar

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B__secedate MIC) 121
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H__exceders_MK281123

H__mezophaewm_KT071036
H__pubescens_KX765792
H__chunense_KX687200
H__margmatuhum_KT071029
H__fiseatum_KY271851
H__monticola_KX765772
H__oreophihum_KY271850
H__clavulipes_KX765771
H__hygophihun_KX765778
H__nigelhm_KX765786
H__gandispomm_KT071023
H__sordescerns_KX765787
H__cistophihun_EU570178
H__celatun_VANFI132B
H__celatum_VANF1132A
H__erebamm_KT218370
H__erebium_KT218321
H__quercetorum_KT218432
H__quercetoram_KT218433
H__celatum_KT218346
H__celatum_KT21$421
H__celatum_KT21845S
H__celatum_KX905038
H__celatum_KT218301
H__celatum_KT218482
H__celatum_KT218490
H__leucozarx_KY081726
H__vehitipes_KT218456
H__bubiferum_KX266254
H__simapizans_KT218484
H__pumuhum_KX765808
H__laterimm_KX687214
H__binws_JF908029
H__cylindiospormm_KX687197
H_adicoaun_KX 765800
H__rsadicomum_FJ168582
H__namum_MK281018
H__nammm_MK280996
H__vesterholtu_FJ943240
H__theobromimun_JX275966
H__odoratissinmun_KX687216
H__naweosum_KX765763
H__sacchaniolens_KX449205
H__genunatom_VANF1116A
H__gewunatum_VANF1116B
H__geminatun_KM390606
H__geminatum_KM390636
H__geminatum_KM390770
H__geminatum_MF039233
H__geminatun_KM39055S
H__alpirmm_KM390650
H__alpirwm_KF309411
H__envtuliniforme_KX657847
B H__salicicola_KM390543
H__salicicola_KM39075S
H__aanenii_KX657845
H__ebumewn_KX657857
H__aanenii_KM390742
H__mins_KM390771
Galerina_pnuinatipes_AJ5S5510

Galenna_pseudocamenina_AJ5$550$

Fic 4. The Bayesian 50% majority rule consensus tree inferred from ITS region. PP> 0.5 are
indicated above branches. Sequences from studied specimens are marked with red colour.

Hebeloma sections are indicated with colours: yellow = Hebeloma, pale blue = Velutipes, dark
blue = Sinapizantia, pink = Scabrispora & Myxocybe, green = Sacchariolentia & Theobromina, and

orange = Denudata. Topology is rooted with Galerina pseudocamerina and G. pruinatipes.

Hebeloma celatum, H. excedens, H. geminatum in Turkey... 131

Our Hebeloma celatum sequences (VANF1132A, B) clustered in
H. sect. Velutipes (PP = 0.99) with H. celatum, H. erebium (Huijsman) Beker
& U. Eberh., and H. quercetorum Quadr. (Fic. 4). This close phylogenetic
relationship can be expected since these species are also morphologically
very similar. Our H. celatum sequences were phylogenetically separated
from representatives retrieved from the database due to nucleotide variations
observed at base 497 (A to G).

Discussion

Hebeloma excedens and Hebeloma geminatum are recorded for the first
time from Turkey, and Hebeloma celatum, previously reported for Turkey by
Grilli & al. (2015), is confirmed for the country.

Hebeloma excedens and H. mesophaeum are not easily separated
from each other because of their close morphological and ecological
similarities. Nonetheless, the differences in pileus morphology are useful for
distinguishing the species. The pileus of H. excedens is less yellow brown
overall and less brown at the disc, and its margin extends over the lamellae.
Also, H. excedens has fibrils on the stipe surface that are not observed in
H. mesophaeum (Cripps & al. 2019, Beker & al. 2016). Our H. excedens
material produced evenly coloured basidiomes that are more slender than
customary for H. mesophaeum and cheilocystidia that are generally swollen
at the base. Molecular data also supported subtle differences between the
two species. Nucleotide variations observed at bases 453 (A to G) and 512
(C to T) support the two species as independent.

Hebeloma geminatum can be confused with H. alpinum and H. aanenii.
Morphologically, the easiest way to separate H. geminatum from close
relatives is through basidiospore and cheilocystidial comparison (Eberhardt
& al. 2015a). The average basidiospore dimensions cited for H geminatum
is <11 um long and 6 um diam. The basidiospores in our collections of
H. geminatum measured 10-11.8 x 5.5-6.5 um, while the cheilocystidia
were generally swollen in the lower half but appeared spathulate. Eberhardt
& al. (2015a) noted that they regarded specimens with cheilocystidial apices
with an average width >9 um to represent H. geminatum; our cheilocystidia
measured <11 um at the apex, meeting their morphological criterion.
Molecular data supported this morphological identification, with nucleotide
variations at bases 140 (G to A), 220 (indel), 562 (A to G) and 565 (G to A)
bases separating H. geminatum from two close relatives.

132 ... Dizkirici, Kalmer, Acar

Grilli & al. (2015) selected a large number of collections from throughout
Europe to infer species diversity and evolutionary history in the two main
Hebeloma sections, Sinapizantia and Velutipes. They included one H. celatum
sample (KT218421) collected from Turkey in 2008 in their study, but as
there was no mention of H. celatum in the Turkish mycobiota since then, we
reevaluated H. celatum through morphological comparisons and molecular
analyses. Hebeloma celatum, which produces mature basidiomes that greatly
resemble those of H. erebium and H. quercetorum, can be distinguished by
its floccose stem and raphanoid odor. More robust basidiomes with larger
stipes help separate H. celatum from H. erebium (Grilli & al. 2015, Cripps
& al. 2019), and the Turkish H. celatum specimens were robust. Likewise,
the clearly clavate and sometimes subcapitate cheilocystidia distinguish our
H. celatum from H. quercetorum, which is characterized by more regularly
ventricose to cylindrical cheilocystidia. Hebeloma celatum associates with
a variety of trees, among which the most commonly recorded is Quercus
(Cripps & al. 2019); our specimens from Turkey were associated with
Quercus species. Sequence variations at bases 140 (A to G) and 475 (A to G)
also support separation of H. celatum from its two close relatives.

The above data well illustrate that macrofungal molecular analyses aid
species identification and that the ITS region is particularly helpful in
understanding the taxonomy and phylogeny of Hebeloma. Two Hebeloma
species, Hebeloma excedens and H. geminatum, have been recorded as new
for Turkey, increasing and the total species from twenty-eight to thirty.
Moreover, our research confirmed the presence of H. celatum in Turkey.

Acknowledgements

This study was financially supported by Van Yiiziincii Yil University (Scientific
Research Project Foundation, FBA-2017-6132), Van, Turkey. We thank the Editor-
in Chief Lorelei L. Norvell and Nomenclature Editor Shaun R. Pennycook for
their critical reading and corrections. We also thank the pre-submission reviewers
Dr Abdullah Kaya (Gazi University, Ankara, Turkey) and Dr Mustafa Emre Akcay
(Van Yiiziincti Yil University, Van, Turkey) for their valuable suggestions.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 135-151
https://doi.org/10.5248/137.135

Ganoderma multipileum and Tomophagus cattienensis—
new records from Pakistan

AISHA UMAR", SHAKIL AHMED’,
LAURA GUZMAN-DAVALOS?, MILAY CABARROI- HERNANDEZ”*

' Department of Botany, University of the Punjab,
Quaid-e-Azam Campus, Lahore, Pakistan

? Department of Botany and Zoology, University of Guadalajara,
Apdo. Postal 1-139, Zapopan, Jal., C.P. 45147, Mexico

* CORRESPONDENCE TO: ash.dr88@gmail.com cabarroi80@gmail.com

ABSTRACT—New records of Ganoderma multipileam and Tomophagus cattienensis collected
from Changa Manga Forest and Lahore, Pakistan, are presented based on morphological and
nuclear rDNA ITS sequence data from fresh basidiomata. Specimens previously treated as
G. lucidum from Pakistan were reviewed and found to represent different species, among
them G. multipileum. Specimens of T. cattienensis determined for the first time from Pakistan
presented morphological features similar to T: colossus but corresponded molecularly to
T. cattienensis; the morphological description for T. cattienensis is expanded accordingly.

KEY WoRDS—Ganodermataceae, morphology, Punjab, taxonomy

Introduction

Ganodermataceae, a monophyletic polypore family with ganodermatoid
basidiospores (Costa-Rezende & al. 2020) previously included in
Polyporaceae (Justo & al. 2017, He & al. 2019), is characterized by its
shelf-like basidiomes, poroid hymenophores, and complex-walled
basidiospores. Amauroderma Murrill, Amaurodermellus Costa-Rezende &
al., Cristataspora Robledo & Costa-Rezende, Foraminispora Robledo & al.,
Furtadoa Costa-Rezende & al., Ganoderma P. Karst., Haddowia Steyaert,
Humphreya Steyaert, and Tomophagus Murrill are some genera currently
included in Ganodermataceae (Murrill 1905; Moncalvo 1996; Moncalvo &

136 ... Umar & al.

Ryvarden 1997; Ryvarden 2004; Kirk & al. 2008; Costa-Rezende & al. 2017,
2020).

Several species of fungi with ganodermatoid basidiospores (specifically
in Ganoderma) have been used as medicine in Asia for more than 2000 years
where they have been cultivated on an industrial scale (Wasser & Weis 1997).
Despite of their significance, this group of fungi remains poorly studied in
many regions of the world such as in Pakistan, where Ganoderma ahmadii
Steyaert, G. applanatum (Pers.) Pat., G. australe (Fr.) Pat., G. boninense Pat.,
G. chalceum (Cooke) Steyaert, G. curtisii (Berk.) Murrill, G. flexipes Pat.,
G. lipsiense (Batsch) G.F. Atk., G. lucidum (Curtis) P. Karst., G. perzonatum
Murrill, G. philippii (Sacc.) Bres., G. praelongum Murrill, G. multicornum
Ryvarden, G. multiplicatum (Mont.) Pat., G. resinaceum Boud., G. tornatum
(Pers.) Bres., G. tsugae Murrill, and Tomophagus colossus (Fr.) Murrill
[= G. colossus (Fr.) C.F. Baker] have been recorded (Ahmad 1956, 1972,
Steyaert 1972, Irshad & al. 2012, Fakhar-ud-Din & Mukhtar 2019). These
species were determined primarily based on morphological concepts;
however, there is much uncertainty regarding which morphological criteria
should be used to assign species to Ganodermataceae and specifically to
Ganoderma (Moncalvo & Ryvarden 1997, Welti & Courtecuisse 2010).

Clarification on the placement of species in Ganoderma or related genera
is essential, not only for taxonomic reasons, but also to investigate the
economical and pharmacological importance of the species that actually
grow in the tropics. The aim of this study was to use morphological and
molecular analyses to show that some specimens previously identified as
G. lucidum in Pakistan actually represent G. multipileum, reported here as
a first record for the country. We also report Tomophagus cattienensis from
Pakistan for the first time.

Materials & methods

Collections & ecology

Fungal specimens were collected in Punjab, Pakistan, during 2018 by the first
author and deposited in the Herbarium of the Institute of Botany, University of the
Punjab, Lahore, Pakistan (LAH). The material was collected in [1] Changa Manga
Forest in the Kasur District, dominated by Dalbergia sissoo DC. and Vachellia nilotica
(L.) PJ.H. Hurter & Mabb. (Fabaceae) and with an average annual rainfall of 1232
mm and average 24 °C temperature (Ahmad & al. 2014) and [2] the New Campus,
University of the Punjab in Lahore, covered by D. sissoo with many rotting trunks and
with an average annual rainfall of 607 mm and average 24 °C temperature (Shirazi &
al. 2019). Additionally, a specimen from XAL was re-examined.

Ganoderma & Tomophagus spp. new for Pakistan ...

TABLE 1. Sequences of Ganoderma, Tomophagus, and Perenniporiella outgroup
included in the analyses. [T] ex-type sequences.

SPECIES

G. ahmadii

G. curtisii

G. leucocontextum

G, lingzhi

G. lucidum

G. martinicense

G. mizoramense

G, multipileum

G. multiplicatum

G. parvulum

VOUCHER/STRAIN

FWP 14329 [T]
CBS 100132
UMNGAI

AY2B

GDGM 40400 [T]
Cui 9166(67)

Dai 12574

K 175217

MUCL 35119

CWN01740
BCRC36123

LIP SW-Mart08-44/
MUCL:GSP44

LIP SW-Mart08-55/
MUCL:GSP55 [T]

UMN-MZ5
UMN-MZ4
CM10
CM110

CWN 04670
Dai 9447
HMAS242384
MN14091107

Dai 13710
Dai 12320
JFL 10004081328

SPC9
SPC5
MUCL 47096

MUCL 53123

URM80765
URM2948

LOCALITY

Pakistan
USA
USA
Pakistan
China
China
China
UK

France

Taiwan
India

Martinique

Martinique

India
India
Pakistan
Pakistan
China
China
China

Myanmar

China
China
China

Brazil
Brazil

Cuba

French Guiana

Brazil

Brazil

GENBANK
ITS1/ITS2

Z37047/ Z37098

JQ781848
MG654117
MN134012
KFO11548
MH109560
KJ143908
KJ14391
MK554779

EU021461
EU021459
KF963257

KF963256

KY643751
KY643750
MW349830
MW349829
KJ143913
KJ143914
JF915409
MK345439

KU572489
K00U572490
MH106879

KU569553
KU569549
MK554783

MK531814

JX310822
JX310821

REFERENCE

Moncalvo & al. 1995
Cao & al. 2012

Loyd & al. 2018
Unpublished

Li & al. 2015
Unpublished

Zhou & al. 2015
Zhou & al. 2015

Cabarroi- Hernandez
&al. 2019

Wang & al. 2009
Wang & al. 2009
Welti & al. 2015

Welti & al. 2015

Crous & al. 2017
Crous & al. 2017
This study

This study

Zhou & al. 2015
Zhou & al. 2015
Wang & al. 2012

Hapuarachchi
& al. 2019

Unpublished
Unpublished

Hapuarachchi
& al. 2018

Bolanos & al. 2016
Bolanos & al. 2016

Cabarroi- Hernandez
&al. 2019

Cabarro0i- Hernandez

&al. 2019
De Lima Jr & al. 2014
De Lima Jr & al. 2014

137

138 ... Umar & al.

GENBANK

SPECIES VOUCHER/STRAIN LOCALITY ITS1/ITS2 REFERENCE

G. resinaceum CBS 194.76 Netherlan0ds KJ143916 Zhou & al. 2015
MUCL 52253 France MK554786 Cabarroi-Hernandez

& al. 2019

G. sichuanense HMAS251146 China JF915401 Wang & al. 2012

G. valesiacum CBS 282.33 UK Z37081/ Z37056 Moncalvo & al. 1995

Ganoderma sp. AU-2019a/HP12 Pakistan MN006955 Unpublished

P. chaquenia MUCL 47648 Argentina FJ411084 Robledo & al. 2009

P. pendula MUCL 47129 Cuba FJ411082 Robledo & al. 2009

T. cattienensis CATPU120 Pakistan MW737424 This study
CATPU121 Pakistan MW737425 This study
CT119 Vietnam JN184398 Le & al. 2012
CT99 [T] Vietnam JN184397 Le & al. 2012

T. colossus 255FL USA MG654427 Loyd & al. 2018
CGMCC5.763 Philippines JQ081068 Wang & al. 2012
UMNFPFL151 USA MG654431 Loyd & al. 2018
URM80450 Brazil JX310825 De Lima Jr & al. 2014
URM83330 Brazil JQ618247 De Lima Jr & al. 2014
TC-02 Vietnam KJ 143923 Zhou & al. 2015

Tomophagus sp. BAB-4989 India KR155077 Unpublished
AUMC 14536 Egypt MW186858 Unpublished

Morphology

Size, shape, and color of basidiomata were noted from fresh material. Color
descriptions follow Munsell (1975). For microscopical analyses, basidiome cross
sections were soaked in 3% KOH, stained with 1% Congo red, and examined under
100x magnification using a Meiji MX4300H compound light microscope. At least
30 basidiospore measurements (face and side view, excluding the apical umbo) were
recorded and rounded to the nearest 0.5 um; dimensions are presented as length
x width (Nagy & al. 2010). Microscopical terms follow, in part, Torres-Torres &
Guzman-Davalos (2012). Color descriptions follow Munsell (1975).

DNA extraction, amplification, and sequencing

Total genomic DNA was extracted from dried specimens following a modified
CTAB procedure (Doyle & Doyle 1987). The ITS1+5.8S+ITS2 rDNA region (ITS) was
amplified using primers ITS1 & ITS2 (White & al. 1990). Reaction mixtures (20 ul)
containing 0.5 ul template DNA, 8.5 ml distilled water, 0.5 ul of each primer, and 10
ml DreamTaqGreen PCR Master Mix (2 X) ran 35 cycles of 95 °C for 30 s, 52 °C for
30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The PCR
amplicons were purified and sequenced by Tsingke Co. Ltd. (Tianjin, China)

Ganoderma & Tomophagus spp. new for Pakistan ... 139

1/99 P. chaquenia MUCL 47548, Argentina
Perenniporiella pendula MUCL 4712, Cuba
Tomophagus sp. BAB-4989, India
T. colossus UMNFL151, USA
Tomophagus sp. AUMC 14536, Egypt

1/98 W
T. colossus URM83330, Brazil T. colossus 2
1/81 N « COIOSSUS F ZI . 4
T. colossus URM80450, Brazil =
cays, | 7: Colossus 255FL, USA )
1/100 T. colossus CGMCCS.763,Philippines E
T. colossus TC-02, Vietnam
T. cattienensis CT99, Vietnam, TYPE
1am T. cattienensis CT119, Vietnam T. cattienlensis

1/89 | _T. cattienensis CATPU120, Pakistan
T. cattienensis CATPU121, Pakistan

Ganoderma lucidum MUCL 35119, France
-/39 | G. lucidum K175217, UK

ie 1/95) G, leucocontextum Code AY2B, Pakistan G. leucocontektum
G. leucocontextum GDGM 40400, China, TYPE
1/100 G. ahmadii FWP 14329,Pakistan
G. valesiacum CBS 282.33, UK
1/100 - G. parvulum MUCL 53123, French Guiana G. parvulum SS.
1/83 G. parvulum MUCL 47096, Cuba
1/100 G. resinaceum MUCL 52253, France
G. resinaceum CBS 194.76, Netherlands
G. lingzhi Dai 12574, China
1/93 |G, sichuanense HMAS251146, China G. sichuanense
1/94 G. lingzhi Cui9166(67), China
0.84/90. G. curtisii UMNGA1, USA
“L. G. curtisii CBS 100132, USA
G. multipileum HMAS242384, China
G. multipileum CWN 04670, China
0.95/75 | | G. lucidum CWNO01740, Taiwan
G. muttipieum CM110, Pakistan G. multipileum
G, multipileum Dai9447, China
G. multipileum CM10, Pakistan
°°" 1 LG. lucidum BCRC36123, India
pe { Ganoderma sp. Code HP12, Pakistan
0.90/77 G. martinicense LIP SW-Mart08-55, Martinique, TYPE
1/7 G. martinicense LIP SW-Mart08-44, Martinique
1/95} G. parvulum URM2948, Brazil
G. parvulum URM80765, Brazil
1/100 G, mizoramense UMN-MZ5, India
G. mizoramense UMN-MZ4, India
99 -— G. multiplicatum SPC9, Brazil G. multiplicatum
G. multiplicatum SPCS, Brazil Brazil
G. multiplicatum MN14091107, Myanmar
he G. multiplicatum Dai 12320, China G, multipli tum
G. multiplicatum Dai 13710, China Asia
G. multiplicatum JFL 14081328, China

G. lucidum s.s.

1/99

G. valesiacum-G. ahmadii

G. resinaceum

1/- <_<
G. curtisii

Ganoderma

1/-

G. martinidense

G. parvulum
0.89/-

G. mizoramens

0.02

Fic. 1. Phylogenetic tree of Ganoderma and Tomophagus based on ITS rDNA sequences generated
by maximum likelihood. Perenniporiella pendula and P. chaquenia were chosen as outgroup.
Posterior probabilities (>0.85) and bootstrap values (>75%) are shown at the branches.

140 ... Umar & al.

Phylogenetic analysis

The data set comprised four DNA sequences from Pakistani material and
45 ITS sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank/)
(TaBLE 1). The ITS data set was subdivided into three parts: ITS1, 5.88, and ITS2.
Perenniporiella chaquenia Robledo & Decock and P. pendula Decock & Ryvarden
were selected as outgroup (Costa-Rezende & al. 2017).

All sequences were automatically aligned with MUSCLE (Edgar 2004) and
manually adjusted using PhyDe® (Miller & al. 2010). JModelTest (Posada & Crandall
1998) was used to determine the best evolutionary model using the corrected
Akaike information criterion (AICc). Maximum Likelihood (ML) analyses were
conducted using RAxML 7.0.4 (Stamatakis 2006) and Bayesian Inference (BI)
analyses were conducted using MrBayes v.3.2.2 (Ronquist & Huelsenbeck 2003). In
the ML analysis, the default priors were used, performing 1000 replicates under the
GTRGAMMA model. BI analyses were run on CIPRES Science Gateway (Miller
& al. 2010). Two independent runs, with 2,000,000 generations each, were carried
out with a sampling frequency every 1000 generations and a burn-in of 25%.
A 50% majority rule consensus tree with posterior probabilities (PP) was obtained.
Convergence of the Markov chains to a stationary distribution was assessed by visual
examination of the log likelihood values in the program Tracer v1.7.1 (Rambaut
& al. 2018). Nodes were considered supported when bootstrap values (BS) were
275% and the PP was 20.85. The final alignments were deposited in TreeBASE
(www.treebase.org).

Phylogenetic results

The evolutionary models that best fit the individual dataset according
to the AICc were ITS] = GTR+G, 5.88 = K80, ITS2 = GTR+G. In BI
analyses, the average standard deviation of split frequencies was 0.007460.
Phylogenetic analysis of the ITS region indicated that our specimens
represent Ganoderma multipileum and Tomophagus cattienensis (Fic. 1).
All sequences of G. multipileum, including the Pakistani collections CM10
and CM110, are supported in one clade (PP = 0.95; BS = 75%). This clade,
which included sequences of specimens from China, India, and Taiwan, also
clustered with sequences from “G. lucidum” specimens and formed a sister
group with G. martinicense Welti & Courtec. and G. parvulum Murrill (0.94
PP; 68% BS). The other two Pakistani sequences (from CATPU120 and
CATPU121) clustered in the strongly supported Tomophagus group (PP = 1;
BS = 100). The Pakistani sequences and the holotype of T! cattienensis (CT99)
formed a monophyletic group with another T. cattienensis sequence from
Vietnam and sequence TC-02 (as T. colossus, surely a misdetermination),
also from Vietnam.

Ganoderma & Tomophagus spp. new for Pakistan ... 141

Taxonomy

A

Fic. 2. Ganoderma multipileum (LAH36825). A. Pileus; B. Pore surface and stipe; C. Section
of context and tubes; D. Basidiospores; E. Cells of crustohymeniderm; F. Basidium. Scale bars:
A-C = 2. cm; D-F = 10 um.

Ganoderma multipileum Ding Hou [as ‘multipilea’],
Quart. J. Taiwan Mus. 3: 101 (1950) FIG: 2

BASIDIOMATA annual; stipitate, solitary, single or with a group of 3-4
pilei growing from the same stipe, light in weight, corky. PILEUs projecting
<12 cm, 12.2 cm wide, and <2.9 cm thick at the base; reniform, dimidiate
to flabelliform, laccate, sulcate, with concentric growing zones; crust thin,
yellowish brown (2.5YR4/8) to brown (10R4/6); MARGIN 0.2-0.3 cm thick,
entire, obtuse, orange-brown (10YR6/8) to brown (5YR 5/8). STIPE 9-9.6 x

142 ... Umar &al.

3-4.1 cm, sub-cylindrical, eccentric to lateral, strongly laccate, woody,
maroon-brown (2.5YR3/6). Pores 6-7 per mm, 110-140 um diam.,
sub-round to round, straw cream (7.5YR8/4) to brown when bruised
(2.5YR4/6). TUBES <1.3 cm long, brown (10YR4/6) to pale brown (2.5YR 8/4).
CONTEXT $1.7 cm thick, with concentric growth zones, brown (7.5YR6/8) to
yellowish-brown (5YR 5/8), with melanoid incrustations.

HyYPHAL SYSTEM DIMITIC: 1) generative hyphae 3.5-5 um diam., thin-walled,
hyaline, clamped; 2) arboriform skeleto-binding hyphae 2.5-7 um diam.,
thick-walled, red-brown to yellow brown. PILEIPELLIS a crustohymeniderm;
cells 15-60 x 4.5-12.5 um, clavate, thick-walled. Basrp1a 20.5-29 x 5.5-8.5
um, claviform, hyaline. Bastp1osporgs 8.0-13.2 x 5.5-7.4 um, ellipsoid to
ovoid, apex truncate, brown, double walled with endosporic ornamentation
as solid, thin, free pillars or column-like projections.

SPECIMENS EXAMINED—PAKISTAN, Punjab, Kasur district, Changa Manga Forest,
31.05°N 73.4072°E, 200 m a.s.L, gregarious on decayed hardwood, Dalbergia sissoo and
Vachellia nilotica, 10 July 2018, Aisha Umar CM10 (LAH36826, GenBank MW349830).
Lahore district, Lahore, New Campus, University of the Punjab, 31.4981°N 73.3044°E,
217 ma.s.l, gregarious on hardwood, on living tree trunk of V. nilotica, 10 April 2018,
Aisha Umar CM110 (LAH36825, GenBank MW349829).

DISTRIBUTION: China, India, Nepal, Pakistan, Philippines, Taiwan, Thailand.

Tomophagus cattienensis X.T. Le & Moncalvo,
Mycol. Prog. 11: 777 (2012) FIG. 3A—-E

BASIDIOMATA annual, sessile, bulky, light in weight, spongy. PILEus
projecting <9.2 cm, 10.2 cm wide, and <7.8 cm thick at the base, dimidiate to
flabelliform, laccate, surface friable, rugose, golden (LOYR7/8), yellow-brown
(10YR8/8), orange-brown (10YR6/8), colors patchy and not on a gradient;
MARGIN 3.4-4 cm, very thick, entire to lobulated, obtuse, rugose, whitish,
white creamy (10YR8/3) to pale yellow brown (10YR7/6). Pores 2-3 (-4) per
mm, angular to round, creamy (7.7Y R8/4) to brown (10YR3/4) when bruised.
TuBESs 0.9-1.2 cm long., golden yellow (10YR7/8). CONTEXT 3.8-7.6 cm
thick, thick at the base, homogeneous, light, soft, creamy white (2.5YR8/4)
when fresh to pale brown (10YR4/6) when dry, powdery.

HYPHAL SYSTEM DIMITIC: 1) generative hyphae 2.5-3 um diam., thin-
walled, clamped, hyaline; 2) skeletal hyphae 2-3 um diam., thick-walled,
hyaline. PILEIPELLIs a crustohymeniderm; cells 40-80 x 7-15 um, narrowly
clavate, thick-walled, apically ornamented. CHLAMYDOSPORES 22-24.5
um diam., globose, double walled, endosporic ornamentation with thick
cylindrical projections, yellowish brown. Basrp1A not seen. BASIDIOSPORES

Ganoderma & Tomophagus spp. new for Pakistan ... 143

Fic. 3. Tomophagus cattienensis (LAH36830). A. Pileus; B. Pore surface and context;
C. Basidiospores; D. Chlamydospores; E. Crustohymeniderm cells. Tomophagus colossus
(XAL-Guzman 35708). F Crustohymeniderm cell; G, H. Basidiospores. Scale bars: A = 2 cm;
B=1 cm; C, D = 20 um.

144 ... Umar & al.

17.4-21.3 x 11.3-14.6 um, ellipsoid to ovoid, apex acute or subacute, or when
collapsed then shortly truncated or even concave, reddish brown, double-
walled with endosporic ornamentation as thick, partially reticulate pillars,
apex hyaline.
SPECIMENS EXAMINED— PAKISTAN, PunyjaB, Lahore district, Lahore, New Campus,
University of the Punjab, 31.4981°N 73.3044°E, 217 m a.s.L, gregarious on hardwood,
on living tree trunk of Dalbergia sissoo, 10 April 2018, Aisha Umar CATPU120
(LAH36830, GenBank MW737424). Kasur district, Changa Manga Forest, 31.05°N
73.4072°E, 200 m a.s.l., gregarious on decayed hardwood, Vachellia nilotica, 10 July
2018, Aisha Umar CATPU121 (LAH36839, GenBank MW737425).
DISTRIBUTION: Pakistan, Vietnam.

ADDITIONAL SPECIMEN EXAMINED (FIG. 3F-H)—Tomophagus colossus: MEXICO,
VERACRUZ, Guzman 35708 (XAL) [as Ganoderma “colossum’].

Discussion

Our morphological and molecular analyses confirm the presence of two
polypores of Ganodermataceae from Pakistan: Ganoderma multipileum and
Tomophagus cattienensis.

For many years, the name Ganoderma lucidum has been misapplied
to Ganoderma specimens from tropical Asia that represent other species
(Wang & al. 2009, Hennicke & al. 2016, Raja & al. 2017). Collections labeled
as ‘G. lucidum’ from world regions outside Europe (where G. lucidum
was described) have appeared in several different lineages in numerous
phylogenetic analyses (Moncalvo & al. 1995, Gottlieb & al. 2000, Smith &
Sivasithamparam 2000, Hong & Jung 2004). Ganoderma multipileum is one
species confused with G. lucidum that is now recognized as representing a
different species (Wang & al. 2009) first described over a half century ago by
Hou (1950).

Loyd & al. (2018) showed that the “multipileum clade” is sister to the
“curtisii clade, with the “multipileum clade” split into two subclades, one
clustering specimens of G. multipileum from China and the other clustering
specimens of G. martinicense from Martinique (type specimen) and from
North America. Ganoderma multipileum, originally described from Taiwan
(Wang & al. 2009), has been recorded from India, China, Nepal, and
Philippines (Wang & al. 2012, Fryssouli & al. 2020). Welti & Courtecuisse
(2010) suggested that G. martinicense might represent a “vicariant from the
Caribbean area’ of G. multipileum. Our phylogeny, although based on a single
DNA marker, agrees with that of Loyd & al. (2018) based on four markers
and supports a relationship between G. multipileum and G. martinicense.

Ganoderma & Tomophagus spp. new for Pakistan ... 145

However, our phylogeny also includes two sequences of G. parvulum
(De Lima Jr & al. 2014) as sister taxon of G. martinicense. Cabarroi-
Hernandez & al. (2019) suggested that the sequences of G. parvulum sensu
De Lima Jr. & al. (2014) might represent G. bibadiostriatum Steyaert, a species
described from Nicaragua (Steyaert 1962) and phylogenetically unrelated to
G. parvulum s.str. (Cabarroi- Hernandez & al. 2019).

Our “multipileum clade” comprises sequences of specimens from China,
India, and Taiwan and CM10 and CM110 (previously labeled G. lucidum)
from Pakistan. The clade also includes the sequences BCRC36123 from India
and CWNO01740 from Taiwan, both referenced at GenBank as G. lucidum but
identified as G. multipileum by Wang & al. (2009), as well as the sequence
Code HP12 referenced at GenBank as Ganoderma sp. from Pakistan (very
probably representing G. multipileum). The ITS tree corroborates our
morphology-based identification and supports extending the distribution of
G. multipileum to Pakistan. Furthermore, we suggest that G. lucidum s.stv. is
not present in that country.

Wang & al. (2009) concluded that, based on morphological characters and
their ITS phylogeny, “G. lucidum” from tropical Asia is divided into two clades,
both of separated from the European G. lucidum s.str. One clade contained
tropical collections (labeled G. multipileum) while a second “unknown” clade
clustered specimens from China and Japan (Wang & al. 2009). Wang & al.
(2012) later recognized the “unknown” clade as G. sichuanense J.D. Zhao &
X.Q. Zhang, while Cao & al. (2012) noted that G. multipileum also occurred
in India and the Philippines.

Ganoderma multipileum is recognized morphologically based on three
features: basidiomes with pilei or with some stipes and pilei growing
together, mostly regular clavate crustohymeniderm cells, and truncated
ellipsoid basidiospores with free fine pillars (Wang & al. 2009, Zhou & al.
2015). According to Cao & al. (2012) G. multipileum inhabits fabaceous
hosts and is distinguished from G. sichuanense (as G. lingzhi Sheng H. Wu
& al.) by its “distinct concentric growth zones in context at maturity, and
finely echinulate basidiospores.” These primary diagnostic features are also
seen in the Pakistani specimens of G. multipileum (Fic. 2). Furthermore,
our specimens were collected growing gregariously on decayed hardwood of
Dalbergia and Vachellia (Fabaceae).

The other Pakistani specimens studied here nested in the Tomophagus
clade. Murrill (1905), who proposed the genus, characterized Tomophagus
by its very lightweight basidiome with a pale soft spongy context and a

146 ... Umar & al.

“labyrinthine” basidiospore surface (Le & al. 2012), as noted in both species
covered here (FIG. 3c,G,H). Tomophagus was typified by Polyporus colossus Fr.
(Murrill 1905). Furtado (1962, 1965), who described the neotype specimen as
having “Ganoderma-type basidiospores,” accepted the species as G. colossus.

Moncalvos (2000) phylogeny revealed an unclassified basal group
composed of G. colossus, G. tsunodae (Lloyd) Sacc. & Trotter, and other taxa.
Subsequent molecular phylogenetic studies confirmed the genus as a well-
established group independent of Ganoderma (Hong & Jung 2004, Le & al.
2012, Xing & al. 2018, He & al. 2019, Costa-Rezende et al 2017, 2020).

Tomophagus cattienensis and T: colossus have been distinguished
primarily by color of the pileal surface and ITS sequence analyses (Le &
al. 2012). Tomophagus cattienensis was distinguished from T!: colossus by
its “red-brown or red-coffeate” (instead of yellow) pileal surface and by its
harder crust and context that “turns pale brown upon drying (instead of
remaining creamy white)” (Le & al. 2012). However, Pakistani specimens
have yellow, yellow-brown, orange-brown pilei and lighter crusts (somewhat
similar to T. colossus or intermediate between the two species) whereas the
color change of the context agrees with that described for T! cattienensis
(Fic. 24). Another character distinguishing T. cattienensis and T. colossus are
the thick-walled crustohymeniderm cells, present in the Pakistani specimens
of T: cattienensis but not previously described for this species. Tomophagus
colossus characteristically has thin-walled cuticular cells (Ryvarden 2000)
observed in the specimen of T: colossus from Mexico (Guzman 35708, XAL)
(Torres-Torres & al. 2015).

Our observations of the Pakistani specimens therefore expand the
morphological description of T’ cattienensis. In our phylogeny, the ITS
sequences of the Pakistani specimens showed only one nucleotide difference
from ITS sequences of the T: cattienensis type specimen in contrast to the
six-nucleotide difference with T. colossus.

Our phylogeny included sequences labelled G. multiplicatum in GenBank,
which separated into two different clades. One clade (PP 1/BS 100) clustered
specimens from China and Myanmar, and the other (PP 1/BS 99) clustered
specimens from Brazil. Morphologically, G. multiplicatum is distinguished
from G. multipileum primarily by its cuticular cells with up to 14 lateral
or apical protuberances (Torres-Torres & Guzman-Davalos 2012, Bolafos
& al. 2016). Originally described from French Guyana, G. multiplicatum
was subsequently found in other parts of South America, Africa, and Asia
(Steyaert 1980, Zhao 1989), including India (Bhosle & al. 2010), Pakistan

Ganoderma & Tomophagus spp. new for Pakistan ... 147

(Fakhar-ud-Din & Mukhtar 2019), and Taiwan (Wang & Wu 2008). The
Asian specimens identified as G. multiplicatum should be re-evaluated and
more DNA regions must be included, to define their taxonomical status.

In this study, we combined macro and microscopic morphological
characters, ecological aspects, as well as molecular data to determine two
species of Ganodermataceae from Pakistan. This is the first report of the
occurrence of G. multipileum and T: cattienensis in this country. Our results
suggest that some species with ganodermatoid basidiospores previously
registered from Pakistan (e.g., G. ahmadii, G. applanatum, G. australe,
G. boninense, G. chalceum, G. curtisii, G. flexipes, G. lipsiense, G. lucidum,
G. praelongum, G. multicornum, G. multiplicatum, G. resinaceum,
G. tornatum, G. tsugae, and T. colossus) need thorough re-evaluation of both
morphology and molecular data before their presence in the country or
taxonomic status can be confirmed.

Acknowledgments

The authors thank Mario Rajchenberg (Centro de Investigacién y Extension
Forestal Andino Patagénico, Chubut, Argentina) and Gerardo Robledo (Universidad
Nacional de Cordoba, Ciudad Universitaria, Argentina) for helpful comments
and their pre-submission expert reviews. MCH gratefully acknowledges financial
support received from CONACYT, Mexico (Postdoc Scholarship). MCH and LGD
thank the University of Guadalajara, Mexico for supporting their research.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2022

January-March 2022— Volume 137, pp. 153-169
https://doi.org/10.5248/137.153

Marasmius tageticolor and M. tucumanus
from the Dominican Republic

Nico.As NIVErIRO’,», NATALIA A. RAM{REZ', CLAUDIO ANGELINI”3

' Instituto de Botanica del Nordeste, IBONE (UNNE-CONICET),
Facultad de Ciencias Exactas y Naturales y Agrimensura,
Universidad Nacional del Nordeste (FACENA-UNNE),
Sargento Cabral 2131, CC 209 Corrientes Capital, CP 3400, Argentina
? Via Cappuccini 78/8, I-33170 Pordenone, Italy
> National Botanical Garden of Santo Domingo,
Santo Domingo, Dominican Republic

“CORRESPONDENCE TO: niconiveiro@gmail.com

ABSTRACT—Two purple Marasmius species from the Dominican Republic are described
and illustrated. Marasmius tageticolor is characterized by its radially striped pileus, distant
lamellae, and elongated spores, and M. tucumanus is characterized by its purple to dark red
pileus, fragile basidioma, and small spores. Both species were sequenced for the first time
and phylogenetically resolved to Marasmius sect. Globulares, closely related to species of
M. ser. Haematocephali + M. ser. Leonini.

Key worps—Agaricales, ITS, Marasmiaceae, mushroom, diversity

Introduction

Marasmius tageticolor and M. tucumanus are members of M. sect.
Globulares (Antonin & Noordeloos 2010, Oliveira & al. 2020), characterized
by the purple coloration of their pileus surface (Singer 1976). Marasmius
tageticolor is a relatively well-known species from the Caribbean and
northern South America, but there are no complete modern descriptions
or publicly available sequences. On the other hand, M. tucumanus has been
found only once, as the type material from northwestern Argentina that has
not been illustrated or sequenced. The aim of this work is to describe and

154 ... Niveiro, Ramirez, Angelini

illustrate both species, evaluate their phylogenetic relationship, and expand
their range of distribution to the Dominican Republic.

Materials & methods

Morphological studies

The specimens, which were collected on litter from man-made lowland
deciduous woods in the Dominican Republic, were photographed and described
macroscopically in situ. The specimens were analyzed macro- and microscopically
following the criteria and terminology proposed by Vellinga (1988) and Lodge & al.
(2004). Color terminology follows Kornerup & Wanscher (1978). For microscopic
analysis, tissue sections were made freehand and mounted in a solution of 5% KOH
(v/w) with 1% phloxine aqueous solution. Melzer’s reagent (Wright & Alberté 2002)
was used to verify amyloidity. The microscopic structures were measured directly
through a 1000x oil-immersion objective or photographs taken with a Leica EC3
built-in camera using ImageJ software (Schneider & al. 2012). The notation “L’ refers
to the number of true lamellae (extending from the stipe insertion to the pileus
margin) counted at the margin of the pileus. The minimum-maximum interval was
provided for the different microscopic structures. For basidiospores, n = number
of spores measured, x = mean spore length x width, Q= length / width ratio, and
Qx = mean Q value. The authors of scientific names agree with Index Fungorum
(2020), and herbarium acronyms follow Thiers (2020). The collected material was
dried at 40 °C and stored in a freezer for a week before being deposited in the Jardin
Botanico Nacional Dr. Rafael M. Moscoso (JBSD) and in the Instituto de Botanica del
Nordeste herbaria (CTES).

DNA extraction, amplification, sequencing

Genomic DNA was extracted from dried specimens implementing a modified
CTAB protocol based on Murray & Thompson (1980). For PCR reactions, we
followed the recommended cycling condition by Mullis & Faloona (1987). Primers
ITSIF and ITS4 (White & al. 1990, Gardes & Bruns 1993) were employed to
amplify the ITS rDNA region. PCR products were checked in 1% agarose gels and
positive reactions were sequenced with one or both PCR primers. DNA extraction,
amplification, and sequencing were performed by Alvalab (Spain). Sequences were
edited using BioEdit 7.2.5 (Hall 1999).

Phylogenetic analysis

The nrITS dataset compiled included our three new sequences and 44 Marasmius
sequences selected from GenBank based on BLAST results and previous studies
(TABLE 1). Crinipellis zonata (Peck) Sacc. was used as outgroup (Aime & Phillips-
Mora 2005). Sequences were aligned with MAFFT 7 (Katoh & Standley 2013) under
the Q-INS-i criteria. The alignment was manually adjusted with MEGA 5 (Tamura
& al. 2011). Potential ambiguously aligned ITS1-ITS2 segments were detected and
deleted using Gblocks 0.91b (Castresana 2000).

Two Marasmius spp. new to the Dominican Republic ...

TABLE 1: Marasmius and outgroup sequences used in the phylogenetic analyses.

Sequences obtained in this study are in bold.

SPECIES

Crinipellis zonata

SERIES

VOUCHER

VPI3355

GENBANK #

AY916692

REFERENCE

Aime &
Phillips-Mora 2005

15S

M. sect. Marasmius

M. rotalis

M. cf. subruforotula

M. tubulatus

Marasmius

Sicciformes

Marasmius

JES145
JES141
JES186
JES192
TYS502
TYS518

KX149000
KX148999
KX149017
KX149018
FJ431280
FJ431279

Shay & al. 2017
Shay & al. 2017
Shay & al. 2017
Shay & al. 2017
Tan & al. 2009
Tan & al. 2009

M. sect. Globulares

M. maximus

M. nivicola

M. pseudo-
purpureostriatus

M. purpureostriatus

M. corrugatiformis

M. cystidiatus

M. ochroleucus

M. strobiluriformis

M. bondoi

M. confertus
var. tenuicystidiatus

M. haematocephalus

M. magnus

M. siccus

M. sullivantii

Globulares

Globulares

Globulares

Globulares

Atrorubentes

Atrorubentes

Atrorubentes

Atrorubentes

Haematocephali

Haematocephali

Haematocephali

Haematocephali

Haematocephali

Haematocephali

KG224
BRNM714571
BRNM714575
BRNM714572
NW286

BRNM7 14566
DED8233
DED8326
1672

CAL1669

LE 295978
BRNM714914
BRNM714915
NW320
BRNM718808

NW434
NW430
ICN179252
FLOR55963
BRNM552709
LE295980
MO218479

FJ904974
FJ904977
FJ904972
FJ904970
EU643513

FJ904978
KX953757
KX953756
MH2160421

MH216191

KF912952

GU266263
GU266264
EU935474
HQ607374

EU935529
EU935535
KX228848
KX228846
HQ607384
KF774130
MK607492

Antonin & al. 2010
Antonin & al. 2010
Antonin & al. 2012
Antonin & al. 2010
Wannathes & al. 2009

Antonin & al. 2010
Shay & al. 2017
Shay & al. 2017

Sharafudheen &
Manimohan 2018

Sharafudheen &
Manimohan 2018

Kiyashko & al. 2014
Antonin & al. 2012
Antonin & al. 2012
Wannathes & al. 2009
Antonin & al. 2012

Wannathes & al. 2009
Wannathes & al. 2009
Magnano & al. 2016
Magnano & al. 2016
Wannathes & al. 2009
Kiyashko & al. 2014

Russell & Grootmyers
(unpub.)

156 ... Niveiro, Ramirez, Angelini

SPECIES SERIES VOUCHER GENBANK # REFERENCE

M. acerosus Leonini TYS427 FJ431214 Tan & al. 2009
TYS458 FJ431213 Tan & al. 2009

M. adhaesus Leonini TYS467 FJ431216 Tan & al. 2009
TYS464 FJ431217 Tan & al. 2009

M. olivascens Leonini TYS424 FJ431266 Tan & al. 2009
TYS426 FJ431265 Tan & al. 2009

M. plicatulus Leonini NW439 EU935480 Wannathes & al. 2009

M. tageticolor Leonini JBSD 130776 MT260146 _‘This paper
JBSD130775 MT260147 This paper

M. tucumanus Leonini JBSD 130778 MT260145_ _— This paper

M. dendrosetosus Spinulosi JES205 KX148995 Shay & al. 2017
JES211 KX148996 Shay & al. 2017

M. longisetosus Spinulosi JO248 JX424040 Oliveira & al. 2014

M. neotrichotus Spinulosi CTES0568167 MF683958 Niveiro & al. 2018

M. nummularius Spinulosi NW266 EU935492 Wannathes & al. 2009
NW396 EU935493 Wannathes & al. 2009

M. paratrichotus Spinulosi DED8248 KX953749 Grace & al. 2019

M. trichotus Spinulosi NW262 EU935490 Wannathes & al. 2009
NW263 EU935491 Wannathes & al. 2009

Phylogenetic reconstruction was inferred using Maximum Likelihood (ML) and
Bayesian Inference (BI). Maximum likelihood was carried out in RAxML-HPC v.8
(Stamatakis 2014), employing the GT[RGAMMA model for the entire dataset. The
analyses first implemented 1000 ML independent searches, each one starting from
one randomized stepwise addition parsimony tree. Only the best scored ML tree
was kept, and node reliability was accessed through nonparametric Bootstrap (BS)
pseudoreplicates under the same model, allowing the program to halt bootstrapping
automatically using the autoMRE option.

Bayesian Inference was performed in MrBayes 3.2.6 (Ronquist & al. 2012). The
evolutionary model for BI was estimated using Akaike Information Criterion (AIC)
as implemented in jModelTest2 v.1.6. (Guindon & Gascuel 2003, Darriba & al. 2012).
We implemented two independent runs, each one beginning from random trees, with
four simultaneous independent chains. A total of 2x10’ generations was carried out,
sampling one tree every 1x10° generation. All phylogenetic analyses were conducted
via CIPRES Science Gateway (Miller & al. 2010). Both analyses (ML and BI) produced
similar topology trees. Only the BI 50% majority-rule consensus tree is shown,
indicating support values Bayesian posterior probabilities (BPP) / rapid bootstrapping
(BS) of each node. A node is considered strongly supported if it showed a BPP 20.98
and/or BS =90%; moderate support is indicated by BPP 20.95 and/or BS =70%.

Two Marasmius spp. new to the Dominican Republic ... 157

0,98/81

1/97

1/75

1/99

1/99

0,95/87

1/-

1/99

1/100

0,99/71

1/97

1/78

M. bondoi EU935474 - SH

M. confertus var. tenuicystidiatus HQ607374 - SH

100 7 ™. strobiluriformis GU266263

M. strobiluriformis GU266264

1/96 M. ochroleucus KF912952
400 [ ™. cystidiatus MH216191

M. cystidiatus MH2160421

ser. Atrorubentes

4/100 M. corrugatiformis KX953756

M. corrugatiformis KX953757

0.99/76 7 M. adhaesus FJ431217

M. adhaesus FJ431216

4/100 M. olivascens FJ431266

1/95
M. olivascens FJ431265

ser. Leonini

M. acerosus FJ431214

1/96
M. acerosus FJ431213

M. purpureostriatus FJ904978

SG

M. pseudopurpureostriatus EU643513

M. siccus.HQ607384

SH

M. siccus KF774130

1/100

sect. Globulares

M. nummularius EU935492

ser. Spinulossi

4/100 M. dendrosetosus KX148995
M. dendrosetosus KX148996
M. longisetosus JX424040

M. haematocephalus EU935535,

SH

M. haematocephalus EU935529

M. tageticolor MT260146 SL

H)

M. tageticolor MT260147 SL

M. tucumanus MT260145 SL

+ ser. Leonini (SL)

M. sullivantii MK607492 - SH

ser. Haematocephalii (S

M. plicatulus EU935480 - SL

1/97

1/99

1/100

1/95

1/97

0,99/100

M. nivicola FJ904970
M. nivicola FJ904972
400 [ M. maximus FJ904974
M. maximus FJ904977

M. magnus KX228846

ser. Globulares (SG)

77 M. neotrichotus MF683958
1/88 M. paratrichotus KX953749
M. trichotus EU935491
4100 M. trichotus EU935490
4/100 M. nummularius EU935493

M. magnus KX228848

4/100 M. rotalis KX149000

M. rotalis KX148999

1/100 M. tubulatus FJ431279

M. tubulatus FJ431280

4/100 M. cf. subruforotula KX149018

sect. Marasmius

M. cf. subruforotula KX149017

Crinipellis zonata AY916692

0.04

Fic. 1: 50% majority-rule consensus tree from Bayesian Inference based on a dataset of nrITS
sequences of Marasmius taxa. Bayesian posterior probability BPP 20.9, and Bootstrap value,

BS =70% are shown.

158 ... Niveiro, Ramirez, Angelini

Phylogenetic results

The nrITS dataset included 48 sequences from 29 Marasmius taxa plus
the outgroup, resulting in an alignment with 728 characters, of which 296 are
parsimony informative.

Our phylogenetic inference (Fic. 1) recovered two major, well-defined
clades: M. sect. Marasmius (BPP = 1; BS = 97%) and M. sect. Globulares
(BPP = 0.93). Within M. sect. Globulares, five moderately to highly
supported subclades are recognized: M. ser. Globulares (BPP = 1; BS = 99%),
M. ser. Atrorubentes (BPP = 0.98; BS = 81%), M. ser. Leonini (BPP = 1; BS = 95%),
M. ser. Spinulosi (BPP = 1; BS =99%) and M. ser. Haematocephali+ M. ser. Leonini
(BPP = 1; BS = 78%).

Our target species, M. tageticolor and M. tucumanus, are sisters (BPP = 0.99;
BS = 71%) and closely related to M. bondoi Wannathes & al. and M. confertus
var. tenuicystidiatus Antonin within the subclade of M. ser. Haematocephali
+ M. ser. Leonini.

Taxonomy

Marasmius tageticolor Berk.,
Hooker’s J. Bot. Kew Gard. Misc. 8: 136. 1856. Fics 2, 3

BASIDIOMATA gregarious, in small groups. PiLEus <20 mm broad,
campanulate to convex when young, then convex to broadly convex,
umbonate or with depressed center, sulcate up to the center in all stages of
development, with slightly crenate edge; surface striped in pigmentation (the
colored stripes correspond to the lamellae and lamellulae lines), center and
radial stripes purple (13E8 “deep magenta’ to 13F8 “dark magenta”) usually
with lighter marginal zones (13C6-7 “greyish magenta’), and white (1A1),
pinkish-white (13A2) to pinkish (13A3) between stripes, brown (7E5) to
dark brown (7F5) with light yellow (4A4) to light orange (5A4) stripes in
dehydrated specimens; dry, subvelutinous, dull. ConTEext thin, whitish
(13A1), odor and taste not distinctive. LAMELLAE free to narrowly adnexed,
broadly ventricose, <5 mm, distant, L = 8-9, white (13A1) at the marginal
edge, pink (13A3) to purplish red (13A6) towards the pileus; usually with
one series of lamellulae between lamellae. Stipe 15-45 x 1-1.5 mm, central,
cylindrical thin, flared upwards, hollow, surface reddish purple brown
(9D7-9E7 “reddish-brown’), lilac (13C7, “purplish red”) towards the apex,
blackish (8F4 “greyish brown to 14F8 “dark purple”) towards the base,
glabrous, dry, with a whitish ochre (5B3-4 “greyish orange”) basal mycelial
patch. ANNULUs absent. SPORE-PRINT not observed.

Two Marasmius spp. new to the Dominican Republic... 159

Fic. 2: Marasmius tageticolor, macroscopic views: a. JBSD 130776; b. JBSD 130775;
c. Detail of basal mycelial patch. Scale bars: a, b = 10 mm. (Photo by C. Angelini).

160 ... Niveiro, Ramirez, Angelini

BASIDIOSPORES 14-21.8 x 3-4(-4.5) um, x = 18.8 x 3.4 um, Q = 3.5-7.1,
Qx = 5.5, n = 25; oblong, acicular to narrowly clavate, with suprahilar
depression, inamyloid, hyaline, smooth, thin-walled. Bastp1a 22-25 x 5-6.5
um, clavate, 4-spored, hyaline, thin-walled. BastproLes 15-25 x 5-6.5 um,
narrowly clavate to fusoid, hyaline, thin-walled. PLEUROCysTIDIA absent.
CHEILOCYSTIDIA in form of Siccus-type broom cells, hyaline, with main
body claviform, 9-15 x 4.5-6 um, setulae <6 um long, and base 1 um diam.
HYMENOPHORAL TRAMA Subregular, hyphae 3-5(-9) um diam., hyaline,
thin-walled, dextrinoid. PILEIPELLIs hymeniform, comprising Siccus-type
broom cells with main body claviform, 9.5-11.5 x 5-7 um, setulae <7 um
long, and bases 1.5 um diam.; hyaline in lighter or discolored interlamellar
region, stramineous in the pigmented region. PILEOCysTrIDIA absent.
STIPITIPELLIS a cutis of smooth hyphae, stramineous, appressed, 3-4 um
diam. CAULOCYSTIDIA absent. CLAMP CONNECTIONS present.

EcoLocy— Forming small groups on dried dicotyledonous twigs and
leaves in the litter.

SPECIMENS EXAMINED—DOMINICAN REPUBLIC: PUERTO PLATA PROVINCE,

Sosua, PUERTO CHIQUITO, on the litter of an anthropized plain forest with broad-leaved

trees, 8/8/2011, leg. C. Angelini 714 (JBSD130775!, GenBank MT260147; CTES!);

5/12/2013, leg. C. Angelini 291 (JBSD130776!, GenBank MT260146; CTES!).
DIsTRIBUTION—Originally described from northwestern Amazonas State,
Brazil (Berkeley 1856); also known from Venezuela, Mexico (Singer 1976),
Panama (Desjardin & Ovrebo 2006), Colombia (Vasco-Palacios & al. 2005),
and USA, Trinidad and Tobago, St. Vincent and the Grenadines (GBIF 2020).
Newly recorded from the Dominican Republic.

COMMENTS—Marasmius tageticolor is characterized by its pileus surface,
which is conspicuously radially striped with a purple-red to purple center
and lamellar stripes, interspersed with lighter interlamellar regions, distant
lamellae, and elongated spores (Singer 1976). The Siccus-type broom cells
of the pileipellis and the absence of pleurocystidia place this species in
M. sect. Globulares (Antonin & Noordeloos 2010) and M. ser. Leonini
(Singer 1976).

A striped pileus is not a frequent feature in M. ser. Leonini (Antonin 2007).
Marasmius poecilus Berk., found in Bolivia, Brazil, and Venezuela, is the closest
species and occasionally considered a synonym (Singer 1965). However, Singer
(1976) highlighted fine differences to separate these two species, such as the
more darkly colored pileus (purplish brown to fulvous with white to yellow
stripes), and longer (35-80 mm) stipe in M. poecilus (Singer 1976).

Two Marasmius spp. new to the Dominican Republic... 161

b C

Bey

Fic. 3: Marasmius tageticolor (JBSD130775).
a. Basidiospores; b. Basidia; c. Basidioles; d. Cheilocystidia. Scale bar = 10 um.

Another striped Neotropical species is M. phaeus Berk. & M.A. Curtis, but
it clearly differs by its dark reddish-brown coloration, lacking a purple hue
(Singer 1976). Some African striped taxa such as M. lilacinoalbus Beeli var.

162 ... Niveiro, Ramirez, Angelini

lilacinoalbus and M. lilacinoalbus var. lilacinocarmineus Singer are similar to
M. tageticolor but differ in their yellowish pileus center and broader spores
(4-5 um; Antonin 2007). Marasmius striipileus Antonin has orange brown
(6C8) to brown (6D8) tints with paler stripes (Antonin 2004), but differs
in the absence of marked streaks with white, yellowish or with pink tints
(Antonin 2007).

Berkeley (1856) described M. tageticolor (based on dehydrated material
and black and white drawings submitted by Spruce from Brazil) as “convex,
membranaceus, umbonate, varying from reddish-brown to deep crimson,
adorned with from eight to ten yellow rays, very minutely wrinkled... Gills
narrow, ventricose, attenuated behind and free, yellow like the rays.” Over
a century later, Singer (1965) reported specimens of M. tageticolor having
brown pileus with deep cream radial stripes and lamellae based on the
type material (Spruce 37 K) and additional material from Bolivia (Singer
B 1981 LIL) that was later identified as M. poecilus (Singer 1976). In that
same work and with additional material from Mexico and Venezuela, Singer
(1976) described M. tageticolor with red pileus and white to ochraceous-
whitish stripes and white lamellae. More recently, Desjardin & Ovrebo
(2006) described M. tageticolor based on specimens from Panama having a
light buff pileus surface with beet red (11-12E-F-8) stripes. The specimens
analyzed here agree with those described by Desjardin & Ovrebo (2006),
but they present some minor differences compared with the protologue
(Berkeley 1856) and descriptions made by Singer (1965, 1976), primarily
regarding the color of the pileus and lamellae. Pigment variations may be
due to different developmental stages, humidity, or observations based
on dehydrated materials in which the lamellae and pileus stripes have
yellowed. Despite these variables, the remaining characteristics agree with
the concept of M. tageticolor. Likewise, the pigmentation of the specimens
studied here and those described by Desjardin & Ovrebo (2006) are similar
to M. haematocephalus var. pseudotageticolor Singer, with which they could
easily be confused, although the M. haematocephalus variety differs by the
presence of pleurocystidia and the insititious or subinsititious stipe base
(Singer & Digilio 1952 as “M. tageticolor”; Singer 1959, 1965, 1976).

Marasmius tucumanus Singer,
Lilloa 25: 206. 1952. Fics 4, 5

BASIDIOMATA solitary or in small groups. PILEUs <15 mm broad,
campanulate to convex with papillate center, striate-sulcate up to the center,

Two Marasmius spp. new to the Dominican Republic ... 163

Fic. 4: Marasmius tucumanus, macroscopic views: a, b. JBSD 130777; c. JBSD130778;
d. Detail of basal mycelial patch. Scale bars: a~c = 10 mm. (Photo by C. Angelini).

164 ... Niveiro, Ramirez, Angelini

with entire to crenate edge, dark purple (11E7, 11F7 “violet brown”) at the
center, lighter towards the margin, from vivid purple red (11A8, “vivid
red”) to dark red (11C8); dry, subvelutinous, sometimes pruinose, mostly
dull or semi-translucent at the margin. CONTEXT thin, whitish (13A1), odor
and taste not distinctive. LAMELLAE free to narrowly adnexed, ventricose,
<2 mm, distant, L = 11-15, white (11A1) at the marginal edge, pink (12A3-4)
towards the pileus; with 3-4 series of lamellulae. StrpE 20-40 x 1.5 mm,
central, cylindrical, equal, hollow, surface purple red (10A8 “vivid red”)
when young, to violet brown (10F8) when mature, glabrous, dry, with a
tomentose, whitish ochre (5B3-4, “greyish orange”) basal mycelial patch.
ANNULUS absent. SPORE-PRINT not observed.

BASIDIOSPORES 10-15 x 3-4.5 um, x = 13.2 x 3.7 um, Q =3.0-4.0, Qx = 3.5,
n= 15; oblong, subclavate, with attenuated suprahilar depression, inamyloid,
hyaline, smooth, thin-walled. Basrp1a 22-27 x 6-7.5 um, clavate, 4-spored,
hyaline, thin-walled. BAsrpIoLeEs 15.5-34.5 x 4-6 um, narrowly clavate to
fusoid, hyaline, thin-walled. PLEUROCysTIDIA absent. CHEILOCYSTIDIA
in form of Siccus-type broom cells, stramineous colored with main body
claviform, 11-19 x 5-9.5 um, setulae <7 um long, and base 1.5 um diam.
HYMENOPHORAL TRAMA Subregular, hyphae 1.5-3.5 um diam., thin-walled,
dextrinoid. PILEIPELLIS hymeniform, comprising Siccus-type broom cells
with main body claviform, 15-22 x 6-7.5 um, hyaline with <7 um long
setulae and base 1.5 um diam. with stramineous coloration. PILEOCYSTIDIA
absent. STIPITIPELLIS an appressed cutis of smooth stramineous hyphae,
3-6 um diam. CAULOCYSTIDIA absent. CLAMP CONNECTIONS present.

EcoLocy—Solitary or forming small groups on dried leaves or small
twigs of dicotyledonous litter.

SPECIMENS EXAMINED—DOMINICAN REPUBLIC, PUERTO PLATA PROVINCE,

Sousa, VERTEDERO, on the litter of an anthropized hill forest with broad-leaved trees,

6/12/2013, leg. C. Angelini 136 (JBSD130777!; CTES!); PUERTO CHIQUITO, on the litter

of an anthropized plane forest with broad-leaved trees, 13/12/2014, leg. C. Angelini 503

(JBSD130778!, GenBank MT260145; CTES!). ARGENTINA, TUuCUMAN PROVINCE,

Tafi, PARQUE ACoNQquyA, 13/03/1951, leg. R. Singer T1460 (holotype, LIL!).
DISTRIBUTION—Previously known only from the type locality, in Argentina

(Singer & Digilio 1952). Newly recorded from the Dominican Republic.

COMMENTS—Marasmius tucumanus is characterized by its relatively fragile
basidiome, purple to dark red coloration, and spores smaller than those of
its most closely related taxa: M. tageticolor or M. haematocephalus complex
(Singer & Digilio 1952). The type specimen (Singer T1460 LIL!) consists of

Two Marasmius spp. new to the Dominican Republic ... 165

a ZNG

W

d

Fic. 5: Marasmius tucumanus (JBSD130777).
a. Basidiospores; b. Basidia; c. Basidioles; d. Cheilocystidia. Scale bar = 10 um.

a single, well-preserved specimen with macroscopic features that agree with
the characters cited in its protologue. Unfortunately, it was not possible to

166 ... Niveiro, Ramirez, Angelini

determine whether the microscopic structures are the same, as destructive
sampling of the type specimen is not allowed.

The presence of purple pigment in the pileus is considered an important
feature by different authors in delimiting groups of species (Singer 1976,
Desjardin & al. 2000, Tan & al. 2009, Wannathes & al. 2009, Shay & al. 2017).
Species with similar pigmentation in M. ser. Leonini are: 1) M. tageticolor,
which differs by its peculiar striped pileus and elongated basidiospores
(Singer 1976, Desjardin & al. 2000); 2) M. amazonicus Henn., known from
Bolivia and Brazil, which is distinguished by its larger (16-58 mm) pileus
with oval to irregularly rounded buff-colored dots (Oliveira & al. 2008);
3) M. purpureotinctus Antonin & P. Roberts, an African species, which
differs by its larger (17.5-20 x 4.5-5.7 um) basidiospores and its pileipellis
with thick-walled broom-cells (Antonin 2013); and 4) M. aratus Massee
[= M. masseei Tkaléec & MeSi¢] from southeastern Asia, which differs by
larger spores (22-32 x 3.5-5 um; Tan & al. 2009). The M. haematocephalus
complex also resembles M. tucumanus in pileal shape and coloration.
However, all its taxa have well differentiated pleurocystidia and larger spores
(14-25 x 3.5-6 um; Singer 1976, Tan & al. 2009, Wannathes & al. 2009,
Shay & al. 2017).

Discussion

Marasmius tageticolor and M. tucumanus are sister species in the
phylogenetic tree (Fic. 1) and closely related to some species belonging to
the traditional M. ser. Haematocephali + M. ser. Leonini. This clade has been
consistently recovered in different papers (Wannathes & al. 2009, Magnano
& al. 2016, Shay & al. 2017, Grace & al. 2019). Among the other species in this
clade included in our analyses, M. bondoi, M. confertus var. tenuicystidiatus,
and M. sullivantii Mont. lacka purple pileus and have pleurocystidia (Antonin
2004, Wannathes & al. 2009), while M. plicatulus Peck has more robust
basidiomata, a dark reddish brown, minutely velutinous pileus surface, and
wider basidiospores (4.8-6.3 um; Desjardin 1987).

Acknowledgments

The authors wish to thank J.J.S. Oliveira (Instituto Nacional de Pesquisas da
Amazonia, Rio de Janeiro) and R.A. Koch (University of Nebraska at Lincoln, USA)
for their critical revision of the manuscript, C. Salvador Montoya for his advice
on phylogenetic analysis, A. Michlig for a preliminary manuscript revision, and
A. Hladky and P. Medina from LIL for photographs of the M. tucumanus type
specimens. CA wishes to thank Ricardo G. Garcia, Francisco Jiménez, Brigido

Two Marasmius spp. new to the Dominican Republic ... 167

Peguero (Jardin Botanico Nacional) and Dr. Rafael M. Moscoso (Santo Domingo,
Dominican Republic) for their interest and encouragement in studying fungi of
the Dominican Republic and for their active cooperation in providing herbarium
material preserved in their institution. NN acknowledges the support of the Consejo
Nacional de Investigaciones Cientificas y Técnicas (CONICET) from Argentina
(PIP 2014-0714), the Secretaria General de Ciencia y Técnica, Universidad Nacional
del Nordeste (SGCyT-UNNE - PI19-P001), and the Agencia Nacional de Promocién
Cientifica y Tecnoldgica (PICT 2016-2529) that made this work possible with their
funding.

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MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. © 2021

January-March 2022— Volume 137, p. 171
https://doi.org/10.5248/137.171

REGIONAL ANNOTATED MYCOBIOTA NEW TO THE MYCOTAXON WEBSITE

ABSTRACT— The 14-page mycobiota reporting on new records of Chaetomium and
Chaetomium-like species on Syagrus coronata from the Caatinga in Bahia, Brazil by
Fortes & Vitéria may now be downloaded from Mycotaxon’s mycobiota webpage.
This well-illustrated range extension, which includes a key to 8 species, brings to 153
the number of free-access fungae uploaded or linked to
http://www.mycotaxon.com/mycobiota/index.html

SOUTH AMERICA
Brazil

NILo GABRIEL SOARES FORTES & NADJA SANTOS VITORIA. New records of
Chaetomium and Chaetomium-like species (Ascomycota, Chaetomiaceae)
on Syagrus coronata from the Raso da Catarina Ecological Station (ESEC),
Caatinga, Bahia, Brazil. 14 p.

ABSTRACT—Studies on the mycobiota associated with the plants of family
Arecaceae are scarce in Brazil, especially in semiarid ecosystems within
the Caatinga domain, which comprises a unique biodiversity. During field
expeditions to the Raso da Catarina Ecological Station, we found six new
records of Chaetomium and Chaetomium-like species for the Caatinga
domain in the State of Bahia. These fungi were colonizing vegetative and
reproductive structures of Syagrus coronata (Mart.) Becc., a palm tree endemic
to the Caatinga and particularly important for animals and people from this
region. We present morphological descriptions, illustrations, comments, and
distribution maps for the fungal species associated with S. coronata.

KEY worps—Arecaceae, biodiversity, fungi, licuri, semiarid, taxonomy

Beltrania shenzhenica sp. nov.
(Zhang & al.— Fie. 2, p. 37)